Identification and characterization of an 8-kDa light chain associated with Dictyostelium discoideum MyoB, a class I myosin

Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an approximately 9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares approximately 30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 microm and underwent a Ca2+-induced change in conformation that increased alpha-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of approximately 0.5 microm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.     

Gastric cancers of Western European and African patients show different patterns of genomic instability

Background

Infection with H. pylori is important in the etiology of gastric cancer. Gastric cancer is infrequent in Africa, despite high frequencies of H. pylori infection, referred to as the African enigma. Variation in environmental and host factors influencing gastric cancer risk between different populations have been reported but little is known about the biological differences between gastric cancers from different geographic locations. We aim to study genomic instability patterns of gastric cancers obtained from patients from United Kingdom (UK) and South Africa (SA), in an attempt to support the African enigma hypothesis at the biological level.

Methods

DNA was isolated from 67 gastric adenocarcinomas, 33 UK patients, 9 Caucasian SA patients and 25 native SA patients. Microsatellite instability and chromosomal instability were analyzed by PCR and microarray comparative genomic hybridization, respectively. Data was analyzed by supervised univariate and multivariate analyses as well as unsupervised hierarchical cluster analysis.

Results

Tumors from Caucasian and native SA patients showed significantly more microsatellite instable tumors (p < 0.05). For the microsatellite stable tumors, geographical origin of the patients correlated with cluster membership, derived from unsupervised hierarchical cluster analysis (p = 0.001). Several chromosomal alterations showed significantly different frequencies in tumors from UK patients and native SA patients, but not between UK and Caucasian SA patients and between native and Caucasian SA patients.

Conclusions

Gastric cancers from SA and UK patients show differences in genetic instability patterns, indicating possible different biological mechanisms in patients from different geographical origin. This is of future clinical relevance for stratification of gastric cancer therapy.

     

Communication impairments in mice lacking Shank1: reduced levels of ultrasonic vocalizations and scent marking behavior

Autism is a neurodevelopmental disorder with a strong genetic component. Core symptoms are abnormal reciprocal social interactions, qualitative impairments in communication, and repetitive and stereotyped patterns of behavior with restricted interests. Candidate genes for autism include the SHANK gene family, as mutations in SHANK2 and SHANK3 have been detected in several autistic individuals. SHANK genes code for a family of scaffolding proteins located in the postsynaptic density of excitatory synapses. To test the hypothesis that a mutation in SHANK1 contributes to the symptoms of autism, we evaluated Shank1(-/-) null mutant mice for behavioral phenotypes with relevance to autism, focusing on social communication. Ultrasonic vocalizations and the deposition of scent marks appear to be two major modes of mouse communication. Our findings revealed evidence for low levels of ultrasonic vocalizations and scent marks in Shank1(-/-) mice as compared to wildtype Shank1(+/+) littermate controls. Shank1(-/-) pups emitted fewer vocalizations than Shank1(+/+) pups when isolated from mother and littermates. In adulthood, genotype affected scent marking behavior in the presence of female urinary pheromones. Adult Shank1(-/-) males deposited fewer scent marks in proximity to female urine than Shank1(+/+) males. Call emission in response to female urinary pheromones also differed between genotypes. Shank1(+/+) mice changed their calling pattern dependent on previous female interactions, while Shank1(-/-) mice were unaffected, indicating a failure of Shank1(-/-) males to learn from a social experience. The reduced levels of ultrasonic vocalizations and scent marking behavior in Shank1(-/-) mice are consistent with a phenotype relevant to social communication deficits in autism.     

p50 (NF-κB1) is an effector protein in the cytotoxic response to DNA methylation damage

The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.     

Unusual repertoire of vocalizations in adult BTBR T+tf/J mice during three types of social encounters

BTBR T+tf/J (BTBR) is an inbred mouse strain that displays social deficits and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. We previously reported an unusual pattern of ultrasonic vocalizations in BTBR pups that may represent a behavioral homolog to the second diagnostic symptom of autism, impaired communication. This study investigated the social and vocal repertoire in adult BTBR mice, to evaluate the role of ultrasonic vocalizations in multiple social situations at the adult stage of development. Three different social contexts were considered: male–female, male–male (resident–intruder) and female–female interactions. Behavioral responses and ultrasonic vocalizations were recorded for BTBR and for the highly social control strain C57BL/6J (B6). No episodes of overt fighting or mating were observed during the short durations of the three different experimental encounters. BTBR displayed lower levels of vocalizations and social investigation in all three social contexts as compared with B6. In addition, the correlation analyses between social investigation and ultrasonic vocalization emission rate showed that in B6 mice, the two variables were positively correlated in all the three different social settings, whereas in BTBR mice, the positive correlation was significant only in the male–female interactions. These findings strongly support the value of simultaneously recording two aspects of the mouse social repertoire: social motivation and bioacoustic communication. Moreover, our findings in adults are consistent with previous results in pups, showing an unusual vocal repertoire in BTBR as compared with B6.     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.     

Quantifying the Evidence for the Risk of Metabolic Syndrome and Its Components following Androgen Deprivation Therapy for Prostate Cancer: A Meta-Analysis

Background

No meta-analysis is yet available for the risk of metabolic syndrome (MetS) following androgen deprivation therapy (ADT) for men with prostate cancer. To summarize the evidence for the link between ADT and MetS or its components quantitatively with a meta-analysis including all studies published to date.

Methods

PubMed and Embase were searched using predefined inclusion criteria to perform meta-analyses on the association between metabolic syndrome, hyperglycemia, diabetes, hypertension, dyslipidemia or obesity and androgen deprivation therapy in patients with prostate cancer. Random effects methods were used to estimate pooled relative risks (RRs) and 95% confidence intervals (CI).

Results

A total of nine studies was included. There was a positive association between ADT and risk of MetS (RR: 1.75 (95% CI: 1.27–2.41)). Diabetes was the only MetS component present in more than 3 studies, and also showed an increased risk following ADT (RR: 1.36 (95% CI: 1.17–1.58)).

Conclusion

This is the first quantitative summary addressing the potential risk of MetS following ADT in men with PCa. The positive RRs indicate that there is a need to further elucidate how type and duration of ADT affect these increased risks of MetS and diabetes as the number of men with PCa treated with ADT is increasing.     

The meaning of significant mean group differences for biomarker discovery

Over the past decade, biomarker discovery has become a key goal in psychiatry to aid in the more reliable diagnosis and prognosis of heterogeneous psychiatric conditions and the development of tailored therapies. Nevertheless, the prevailing statistical approach is still the mean group comparison between “cases” and “controls,” which tends to ignore within-group variability. In this educational article, we used empirical data simulations to investigate how effect size, sample size, and the shape of distributions impact the interpretation of mean group differences for biomarker discovery. We then applied these statistical criteria to evaluate biomarker discovery in one area of psychiatric research—autism research. Across the most influential areas of autism research, effect size estimates ranged from small (d = 0.21, anatomical structure) to medium (d = 0.36 electrophysiology, d = 0.5, eye-tracking) to large (d = 1.1 theory of mind). We show that in normal distributions, this translates to approximately 45% to 63% of cases performing within 1 standard deviation (SD) of the typical range, i.e., they do not have a deficit/atypicality in a statistical sense. For a measure to have diagnostic utility as defined by 80% sensitivity and 80% specificity, Cohen’s d of 1.66 is required, with still 40% of cases falling within 1 SD. However, in both normal and nonnormal distributions, 1 (skewness) or 2 (platykurtic, bimodal) biologically plausible subgroups may exist despite small or even nonsignificant mean group differences. This conclusion drastically contrasts the way mean group differences are frequently reported. Over 95% of studies omitted the “on average” when summarising their findings in their abstracts (“autistic people have deficits in X”), which can be misleading as it implies that the group-level difference applies to all individuals in that group. We outline practical approaches and steps for researchers to explore mean group comparisons for the discovery of stratification biomarkers.     

Bryophyte biomass and species richness on the Park Grass Experiment,Rothamsted, UK

The relationships between bryophyte biomass and species richness and soil pH, nutrient applications and vascular plant biomass and species richness were analyzed for the Park Grass Experiment (Rothamsted, UK). The study examined the abundance of bryophytes in relation to long-term fertilizer and lime application and to fertilizer treatments recently being ceased on some plots. The probability of bryophytes being present on a plot increased with increasing soil pH, and on plots at soil pH 3.3–4.5, the lowest values in this experiment, there were virtually no mosses present. Total bryophyte biomass decreased with increasing vascular plant biomass and vascular plant richness. Both bryophyte biomass and species richness showed a curvilinear response to soil pH. Bryophyte biomass was markedly increased on plots where nitrogen (N) fertilization had recently been ceased. The abundance of the common bryophyte species showed individualistic responses to treatments. N had a negative effect on the abundance of Brachythecium rutabulum. Increasing soil pH, and the application of phosphorus (P) and potassium (K) fertilizer together, had a positive effect on Eurhynchium praelongum. This species was also negatively affected by N, but tolerated larger amounts of it (100–150 kg ha^–1 N) than B. rutabulum. An ephemeral moss, Bryum subapiculatum, had a unimodal response to soil pH but showed no response to N, P, K or other explanatory variables.