Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.

Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response.

Caenorhabditis elegans dnj-14, the orthologue of the DNAJC5 gene mutated in adult onset neuronal ceroid lipofuscinosis, provides a new platform for neuroprotective drug screening and identifies a SIR-2.1-independent action of resveratrol.

Adult onset neuronal lipofuscinosis (ANCL) is a human neurodegenerative disorder characterized by progressive neuronal dysfunction and premature death. Recently, the mutations that cause ANCL were mapped to the DNAJC5 gene, which encodes cysteine string protein alpha. We show here that mutating dnj-14, the Caenorhabditis elegans orthologue of DNAJC5, results in shortened lifespan and a small impairment of locomotion and neurotransmission. Mutant dnj-14 worms also exhibited age-dependent neurodegeneration of sensory neurons, which was preceded by severe progressive chemosensory defects. A focussed chemical screen revealed that resveratrol could ameliorate dnj-14 mutant phenotypes, an effect mimicked by the cAMP phosphodiesterase inhibitor, rolipram. In contrast to other worm neurodegeneration models, activation of the Sirtuin, SIR-2.1, was not required, as sir-2.1; dnj-14 double mutants showed full lifespan rescue by resveratrol. The Sirtuin-independent neuroprotective action of resveratrol revealed here suggests potential therapeutic applications for ANCL and possibly other human neurodegenerative diseases.

Regulation of base excision repair proteins by ubiquitylation.

Human cellular DNA is under constant attack from both endogenous and exogenous mutagens, and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying complexity. BER thus maintains genome stability, and prevents the development of human diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.

A systematic analysis of protein palmitoylation in Caenorhabditis elegans.

BACKGROUND: Palmitoylation is a reversible post-translational protein modification which involves the addition of palmitate to cysteine residues. Palmitoylation is catalysed by the DHHC family of palmitoyl-acyl transferases (PATs) and reversibility is conferred by palmitoyl-protein thioesterases (PPTs). Mutations in genes encoding both classes of enzymes are associated with human diseases, notably neurological disorders, underlining their importance. Despite the pivotal role of yeast studies in discovering PATs, palmitoylation has not been studied in the key animal model Caenorhabditis elegans. RESULTS: Analysis of the C. elegans genome identified fifteen PATs, using the DHHC cysteine-rich domain, and two PPTs, by homology. The twelve uncategorised PATs were officially named using a dhhc-x system. Genomic data on these palmitoylation enzymes and those in yeast, Drosophila and humans was collated and analysed to predict properties and relationships in C. elegans. All available C. elegans strains containing a mutation in a palmitoylation enzyme were analysed and a complete library of RNA interference (RNAi) feeding plasmids against PAT or PPT genes was generated. To test for possible redundancy, double RNAi was performed against selected closely related PATs and both PPTs. Animals were screened for phenotypes including size, longevity and sensory and motor neuronal functions. Although some significant differences were observed with individual mutants or RNAi treatment, in general there was little impact on these phenotypes, suggesting that genetic buffering exists within the palmitoylation network in worms. CONCLUSIONS: This study reports the first characterisation of palmitoylation in C. elegans using both in silico and in vivo approaches, and opens up this key model organism for further detailed study of palmitoylation in future.

Reproductive and margin of safety of a fixed-dose combination injectable endectocide (0.2 mg/kg doramectin; 6.0 mg/kg levamisole hydrochloride) in cattle.

We present a fixed-dose combination injectable (FDCI) solution for cattle formulated for a single subcutaneous administration at a dose rate of 1 ml/25 kg of body weight to deliver a dose of 0.2 mg/kg of doramectin and 6.0 mg/kg of levamisole hydrochloride (5.1 mg/kg base equivalent). This drug product is marketed in the United States under the tradename Valcor® and in Australia and New Zealand under the tradename Dectomax V®. Both levamisole and doramectin have histories of safe and effective use in ruminants, with safety margins of 3X and 25X, respectively. Three studies were conducted to demonstrate the safety of the new FDCI: margin of safety (Study 1), and reproductive safety in sexually nulliparous beef heifers (Studies 2 and 3). In Study 1, 3-month-old sexually intact male and female calves were given either saline (control) or 1X, 2X, or 3X FDCI on Days 0, 14, and 28. General health, clinical, and neurological observations were made throughout the study, and clinical and pathology evaluations were made at study end. Studies 2 and 3 demonstrated the reproductive safety of the FDCI on sexually nulliparous beef heifers using estrus synchronization and timed artificial insemination. Treatments of either saline (control) or 3X FDCI were administered to coincide with either folliculogenesis, implantation, organogenesis, early gestation, or late gestation. Reproductive safety was demonstrated by evaluating rates of conception, calving, abortion, and stillbirth, dystocia scores, and calf health. In all studies, the FDCI at 1X, 2X, or 3X dosages was well tolerated. In the margin of safety study, 3X calves showed increased incidence of salivation for up to 8 h post-dosing compared to other groups. Injection sites were palpable post-dosing in all three FDCI groups but resolved by Day 28 in all but one animal each in 2X and 3X. In the reproductive safety studies, the FDCI had no effect on conception, pregnancy, fetal development, or postnatal viability. Injection site swelling was increased in frequency and duration compared to controls. The studies demonstrate the safety of the new FDCI in cattle from 3 months of age and in reproducing heifers during all reproductive stages from folliculogenesis through gestation and up to a month post-partum.

Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting.

A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms.

Synovex ONE Grower improves growth performance for at least 200 days in growing beef steers and heifers fed in confinement for slaughter.

Growth-promoting implants are broadly used in the feedlot industry to improve growth performance and to increase production efficiencies. With cattle being fed longer and to heavier weights, there is demand for extended-release implants that payout for at least 200 d. Our objective was to evaluate feedlot growth of Synovex ONE Grower, a moderate potency (150 mg trenbolone acetate [TBA] and 21 mg estradiol benzoate [EB]), extended-release, growth-promoting implant for 200 d. At four locations (Texas, Idaho, California, and Nebraska), 200 steers (n = 800; d 0 body weight [BW] = 320.2 ± 9.5 kg) and 200 heifers (n = 800; d 0 BW = 311.5 ± 9.5 kg) were blocked by BW and randomized to 1 of 2 treatments: 1) Control, empty subcutaneous needle inserted and extracted from the middle third of one ear; 2) ONE Grower, 150 mg TBA and 21 mg EB extended-release implant administered in middle third of one ear. Treatments were commingled within pen of the same sex (n = 4/site; 2/sex/site) in a split plot design replicated across four sites. Cattle were fed finishing ration ad libitum common to each geographical region at least once daily and were observed for any abnormal health events twice daily. Treatments were administered on d 0. Mid-study implant site evaluations were performed on d 35 or 41. Initial BW was recorded on d 0 and final BW was recorded on d 200 to 204. Cattle were harvested from d 201 to 231; however, carcass data were not collected due to slaughter facility complications brought on by the COVID-19 pandemic. Data were analyzed using the PROC MIXED and PROC GLIMMIX procedures of SAS (Version 9.4, SAS Institute, Cary, NC; P < 0.05), and animal was the experimental unit. There were no treatment × sex interactions (P ≥ 0.052) for any variable. Final BW on d 200 was greater (P < 0.01) for steers and heifers implanted with ONE Grower compared to Control; ONE Grower improved final BW by 5.7% for steers and 3.9% for heifers. Overall average daily gain (ADG) from d 0 to 200 was greater (P < 0.01) for ONE Grower steers and heifers compared to Control with an increase in ADG of 13.1% for steers and 8.9% for heifers. For cattle implanted with ONE Grower, implant retention rates at d 35 or 41 were 95.7% and 96.3% for steers and heifers, respectively. There was no difference (P ≥ 0.32) in percentage deads, removals, or bullers (steers) between treatments. Synovex ONE Grower improved final BW and ADG in feedlot steers and heifers fed for at least 200 d.

Innate and acquired immune responses of colostrum-fed neonatal Holstein calves following intranasal vaccination with two commercially available modified-live virus vaccines.

OBJECTIVE: To compare immune responses induced by 2 commercially available vaccines with a bovine herpesvirus type 1 (BHV1) component following intranasal (IN) administration to colostrum-fed calves. ANIMALS: 90 male Holstein calves (ages, 5 to 14 days). PROCEDURES: In a randomized complete block design, each calf received 2 mL (1 mL/nostril) of vaccine A (n = 30), vaccine B (30), or saline (0.9% NaCl) solution (30) on day 0. Blood samples were collected for determination of serum anti-BHV1 IgG titer, and nasal fluid (NF) samples were collected for determination of interferon (IFN)-α and IFN-γ concentrations and for secretory IgA titers against BHV1, Mannheimia haemolytica, and Pasteurella multocida at predetermined times for 42 days after vaccination. RESULTS: All calves were seropositive for anti-BHV1 IgG, and the mean anti-BHV1 IgG titer did not differ significantly among the 3 groups at any time. Both vaccines induced significant transient increases in NF IFN-α and IFN-γ concentrations. On day 5, mean IFN-α concentration and the proportion of calves with detectable IFN-α concentrations for the vaccine A group were significantly greater than those for the vaccine B and control groups. On day 42, the mean NF anti-P multocida IgA titers for both vaccine groups were significantly greater than that of the control group. CONCLUSIONS AND CLINICAL RELEVANCE: Both vaccines induced innate and acquired immune responses in calves with colostral antibodies. The magnitude of the IFN-α response and proportion of calves with detectable IFN-α differed between the 2 vaccine groups. Both vaccines appeared to enhance the IgA response against P multocida.

Analysis of the brain palmitoyl-proteome using both acyl-biotin exchange and acyl-resin-assisted capture methods.

Palmitoylation is a reversible post-translational protein modification in which palmitic acid is added to cysteine residues, allowing association with different cellular membranes and subdomains. Recently, techniques have been developed to identify palmitoylation on a proteome-wide scale in order to reveal the full cellular complement of palmitoylated proteins. However, in the studies reported to date, there is considerable variation between the sets of identified palmitoyl-proteins and so there remains some uncertainty over what constitutes the definitive complement of palmitoylated proteins even in well-studied tissues such as brain. To address this issue, we used both acyl-biotin exchange and acyl-resin-assisted capture approaches using rat brain as a common protein source. The palmitoyl-proteins identified from each method by mass spectrometry were then compared with each other and previously published studies. There was generally good agreement between the two methods, although many identifications were unique to one method, indicating that at least some of the variability in published palmitoyl proteomes is due to methodological differences. By combining our new data with previous publications using mammalian cells/tissues, we propose a high confidence set of bona fide palmitoylated proteins in brain and provide a resource to help researchers prioritise candidate palmitoyl-proteins for investigation.

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis.

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.

Chemotherapy with gemcitabine-containing regimens for locally recurrent or metastatic nasopharyngeal carcinoma.

BACKGROUND: Results from Phase II trials conducted in Asia have shown that gemcitabine alone (GEM) or with cisplatin (GC) is active among patients with metastatic or locally recurrent nasopharyngeal carcinoma (NPC). METHODS: At the Princess Margaret Hospital (PMH), Toronto, 32 patients with NPC were treated with GEM (n = 18) or GC (n = 14) from January 2000 to October 2001. Patients either received 1000 mg/m(2) GEM on Days 1, 8, and 15 every 28 days as a single agent, or with cisplatin (CG) given on day 2 at 70 mg/m(2). RESULTS: Most patients (91%) were of Southeast Asian ancestry and 29 (91%) had Type 2 (World Health Organization 1991 classification) nonkeratinizing histology. Sixteen of the GEM (89%) and five (36%) of the GC patients had received chemotherapy before entering the study. Median follow-up was 32 weeks (range, 2-97 weeks) for both groups. In the GEM group, there were five (28%) partial responses (PR) and one (6%) complete response (CR), giving an overall response rate of 34% (95% confidence interval [CI], 13.59). In the GC group, there were two (14%) CRs and seven PRs (50%), giving an overall response of 64% (95% CI, 35-87). Hematologic toxicity was dose limiting but uncomplicated. Nonhematologic toxicity included one patient with reversible reactivation of hepatitis, one with Grade 3 cisplatin-related sensory neuropathy, and three with cardiovascular events that were possibly related to chemotherapy. The median duration of response for the GEM and GC patients was 17 and 24 weeks and the 1-year survival rate was 48% (95% CI, 18-78) and 69% (95% CI, 40-99), respectively. Median survival has not been reached. CONCLUSIONS: Our study confirms that GEM is an active and tolerable drug for patients with NPC.

Infection outcome and cytokine gene expression in Brugia pahangi- infected gerbils (Meriones unguiculatus) sensitized with Brucella abortus.

Filarial infections have been associated with the development of a strongly polarized Th2 host immune response and a severe impairment of mitogen-driven proliferation and type 1 cytokine production in mice and humans. The role of this polarization in the development of the broad spectra of clinical manifestations of lymphatic filariasis is still unknown. Recently, data gathered from humans as well as from immunocompromised mouse models suggest that filariasis elicits a complex host immune response involving both Th1 and Th2 components. However, responses of a similar nature have not been reported in immunologically intact permissive models of Brugia infection. Brucella abortus-killed S19 was inoculated into the Brugia-permissive gerbil host to induce gamma interferon (IFN-gamma) production. Gerbils were then infected with B. pahangi, and the effect of the polarized Th1 responses on worm establishment and host cellular response was measured. Animals infected with both B. abortus and B. pahangi showed increased IFN-gamma and interleukin-10 (IL-10) and decreased IL-4 and IL-5 mRNA levels compared with those in animals infected with B. pahangi alone. These data suggest that the prior sensitization with B. abortus may induce a down regulation of the Th2 response associated with Brugia infection. This reduced Th2 response was associated with a reduced eosinophilia and an increased neutrophilia in the peritoneal exudate cells. The changes in cytokine and cellular environment did not inhibit the establishment of B. pahangi intraperitoneally. The data presented here suggest a complex relationship between the host immune response and parasite establishment and survival that cannot be simply ascribed to the Th1/Th2 paradigm.