EcoGenoRisk: Developing a computational ecological risk assessment tool for synthetic biology.

The expanding field of synthetic biology (synbio) supports new opportunities in the design of targeted bioproducts or modified microorganisms. However, this rapid development of synbio products raises concerns surrounding the potential risks of modified microorganisms contaminating unintended environments. These potential invasion risks require new bioinformatic tools to inform the design phase. EcoGenoRisk is a newly constructed computational risk assessment tool for invasiveness that aims to predict where synbio microorganisms may establish a population by screening for habitats of genetically similar microorganisms. The first module of the tool identifies genetically similar microorganisms and potential ecological relationships such as competition, mutualism, and inhibition. In total, 520 archaeal and 32,828 bacterial complete assembly genomes were analyzed to test the specificity and accuracy of the tool as well as to characterize the enzymatic profiles of different taxonomic lineages. Additionally, ecological relationships were analyzed to determine which would result in the greatest potential overlap between shared functional profiles. Notably, competition displayed the significantly highest overlap of shared functions between compared genomes. Overall, EcoGenoRisk is a flexible software pipeline that assists environmental risk assessors to query large databases of known microorganisms and prioritize follow-up bench scale studies.

NOS-dependent effects of plantar mechanical stimulation on mechanical characteristics and cytoskeletal proteins in rat soleus muscle during hindlimb suspension.

The study was aimed at investigating the mechanisms and structures which determine mechanical properties of skeletal muscles under gravitational unloading and plantar mechanical stimulation (PMS). We hypothesized that PMS would increase NO production and prevent an unloading-induced reduction in skeletal muscle passive stiffness. Wistar rats were hindlimb suspended and subjected to a daily PMS and one group of stimulated animals was also treated with nitric oxide synthase (NOS) inhibitor (L-NAME). Animals received mechanical stimulation of the feet for 4 h a day throughout 7-day hindlimb suspension (HS) according to a scheme that mimics the normal walking of the animal. Seven-day HS led to a significant reduction in soleus muscle weight by 25%. However, PMS did not prevent the atrophic effect induced by HS. Gravitational unloading led to a significant decrease in maximum isometric force and passive stiffness by 38% and 31%, respectively. The use of PMS prevented a decrease in the maximum isometric strength of the soleus muscle. At the same time, the passive stiffness of the soleus in the PMS group significantly exceeded the control values by 40%. L-NAME (NOS inhibitor) administration attenuated the effect of PMS on passive stiffness and maximum force of the soleus muscle. The content of the studied cytoskeletal proteins (α-actinin-2, α-actinin-3, desmin, titin, nebulin) decreased after 7-day HS, but this decrease was successfully prevented by PMS in a NOS-dependent manner. We also observed significant decreases in mRNA expression levels of α-actinin-2, desmin, and titin after HS, which was prevented by PMS. The study also revealed a significant NOS-dependent effect of PMS on the content of collagen-1a, but not collagen-3a. Thus, PMS during mechanical unloading is able to maintain soleus muscle passive tension and force as well as mRNA transcription and protein contents of cytoskeletal proteins in a NOS-dependent manner.

Predominant synthesis of giant myofibrillar proteins in striated muscles of the long-tailed ground squirrel Urocitellus undulatus during interbout arousal.

Molecular mechanisms underlying muscle-mass retention during hibernation have been extensively discussed in recent years. This work tested the assumption that protein synthesis hyperactivation during interbout arousal of the long-tailed ground squirrel Urocitellus undulatus should be accompanied by increased calpain-1 activity in striated muscles. Calpain-1 is known to be autolysed and activated in parallel. Western blotting detected increased amounts of autolysed calpain-1 fragments in the heart (1.54-fold, p < 0.05) and m. longissimus dorsi (1.8-fold, p < 0.01) of ground squirrels during interbout arousal. The total protein synthesis rate determined by SUnSET declined 3.67-fold in the heart (p < 0.01) and 2.96-fold in m. longissimus dorsi (p < 0.01) during interbout arousal. The synthesis rates of calpain-1 substrates nebulin and titin in muscles did not differ during interbout arousal from those in active summer animals. A recovery of the volume of m. longissimus dorsi muscle fibres, a trend towards a heart-muscle mass increase and a restoration of the normal titin content (reduced in the muscles during hibernation) were observed. The results indicate that hyperactivation of calpain-1 in striated muscles of long-tailed ground squirrels during interbout arousal is accompanied by predominant synthesis of giant sarcomeric cytoskeleton proteins. These changes may contribute to muscle mass retention during hibernation.

Effect of L-Arginine on Titin Expression in Rat Soleus Muscle After Hindlimb Unloading.

Nitric oxide (NO), produced by NO-synthases via L-arginine oxidation, is an essential trigger for signaling processes involved in structural and metabolic changes in muscle fibers. Recently, it was shown that L-arginine administration prevented the decrease in levels of the muscle cytoskeletal proteins, desmin and dystrophin, in rat soleus muscle after 14 days of hindlimb unloading. Therefore, in this study, we investigated the effect of L-arginine administration on the degree of atrophy changes in the rat soleus muscles under unloading conditions, and on the content, gene expression, and phosphorylation level of titin, the giant protein of striated muscles, able to form a third type of myofilaments-elastic filaments. A 7-day gravitational unloading [hindlimb suspension (HS) group] resulted in a decrease in the soleus weight:body weight ratio (by 31.8%, p < 0.05), indicating muscle atrophy development. The content of intact titin (T1) decreased (by 22.4%, p < 0.05) and the content of proteolytic fragments of titin (T2) increased (by 66.7%, p < 0.05) in the soleus muscle of HS rats, compared to control rats. The titin gene expression and phosphorylation level of titin between these two groups were not significantly different. L-Arginine administration under 7-day gravitational unloading decreased the degree of atrophy changes and also prevented the decrease in levels of T1 in the soleus muscle as compared to HS group. Furthermore, L-arginine administration under unloading resulted in increased titin mRNA level (by 76%, p < 0.05) and decreased phosphorylation level of T2 (by 28%, p < 0.05), compared to those in the HS group. These results suggest that administration of L-arginine, the NO precursor, under unloading decreased the degree of atrophy changes, increased gene expression of titin and prevented the decrease in levels of T1 in the rat soleus muscle. The results can be used to search for approaches to reduce the development of negative changes caused by gravitational unloading in the muscle.

Cellular and molecular signatures of alcohol-induced myopathy in women.

Alcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1) both fast and slow muscle fibers are subjected to atrophy; 2) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content.

Increased Autolysis of µ-Calpain in Skeletal Muscles of Chronic Alcohol-Fed Rats.

BACKGROUND: Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of µ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in µ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake. METHODS: SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine µ-calpain autolysis as well as µ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR. RESULTS: The amounts of the autolysed isoform (78 kDa) of full-length µ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats. CONCLUSIONS: In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of µ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

Isoform composition and gene expression of thick and thin filament proteins in striated muscles of mice after 30-day space flight.

Changes in isoform composition, gene expression of titin and nebulin, and isoform composition of myosin heavy chains as well as changes in titin phosphorylation level in skeletal (m. gastrocnemius, m. tibialis anterior, and m. psoas) and cardiac muscles of mice were studied after a 30-day-long space flight onboard the Russian spacecraft "BION-M" number 1. A muscle fibre-type shift from slow-to-fast and a decrease in the content of titin and nebulin in the skeletal muscles of animals from "Flight" group was found. Using Pro-Q Diamond staining, an ~3-fold increase in the phosphorylation level of titin in m. gastrocnemius of mice from the "Flight" group was detected. The content of titin and its phosphorylation level in the cardiac muscle of mice from "Flight" and "Control" groups did not differ; nevertheless an increase (2.2 times) in titin gene expression in the myocardium of flight animals was found. The observed changes are discussed in the context of their role in the contractile activity of striated muscles of mice under conditions of weightlessness.

Comorbid medical illness in bipolar disorder.

BACKGROUND: Individuals with a mental health disorder appear to be at increased risk of medical illness. AIMS: To examine rates of medical illnesses in patients with bipolar disorder (n = 1720) and to examine the clinical course of the bipolar illness according to lifetime medical illness burden. METHOD: Participants recruited within the UK were asked about the lifetime occurrence of 20 medical illnesses, interviewed using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) and diagnosed according to DSM-IV criteria. RESULTS: We found significantly increased rates of several medical illnesses in our bipolar sample. A high medical illness burden was associated with a history of anxiety disorder, rapid cycling mood episodes, suicide attempts and mood episodes with a typically acute onset. CONCLUSIONS: Bipolar disorder is associated with high rates of medical illness. This comorbidity needs to be taken into account by services in order to improve outcomes for patients with bipolar disorder and also in research investigating the aetiology of affective disorder where shared biological pathways may play a role.

COASTAL DIATOM-ENVIRONMENT RELATIONSHIPS IN THE BRACKISH BALTIC SEA(1).

High-quality calibration data sets are required when diatom assemblages are used for monitoring ecological change or reconstructing palaeo-environments. The quality of such data sets can be validated, in addition to other criteria, by the percentage of significant unimodal species responses as a measure of the length of an environmental gradient. This study presents diatom-environment relationships analyzed from a robust data set of diatom communities living on submerged stones along a 2,000 km long coastline in the Baltic Sea area, including 524 samples taken at 135 sites and covering a salinity gradient from 0.4 to 11.4. Altogether, 487 diatom taxa belonging to 102 genera were recorded. Detrended canonical correspondence analysis showed that salinity was the overriding environmental factor regulating diatom community composition, while exposure to wave action and nutrient concentrations were of secondary importance. Modeling the abundances of the 58 most common diatom taxa yielded significant relationships with salinity for 57 taxa. Twenty-three taxa showing monotonic responses were species with optimum distributions in freshwater or marine waters. Thirty-four taxa showing unimodal responses were brackish-water species with maximum distributions at different salinities. Separate analyses for small (cell biovolume <1,000 µm(3) ) and large (≥1,000 µm(3) ) taxa yielded similar results. In previous studies along shorter salinity gradients, large and small epilithic diatom taxa responded differently. From our large data, we conclude that counts of large diatom taxa alone seem sufficient for indicating salinity changes in coastal environments with high precision.