As we collect soil metagenome sequence data, we need to improve how we mine such datasets. For example, the way we conduct BLAST searches might overlook valuable information, while the unassembled reads might be too short for annotating genes with confidence. Thus, we might well need to develop new assembly and annotation algorithms. Another challenge is how to integrate different kinds of omics data, including metatranscriptomics and metaproteomics, to better understand functional processes of soil microbial communities. Metagenome sequence data, while informative, provides information about genes with the potential for being expressed, but cannot determine which ones are functional. Also, because we sequence total DNA, it is not possible to distinguish genes from actively growing cells from those in dormant or dead cells. Perhaps some analyses should be reserved for that fraction of DNA from active community members—for example, by extracting DNA that is allowed to incorporate stable isotopes or bromodeoxyuridine during replication. Ultimately,large pots plastic combining these approaches should enable us to gain a better understanding of which microbes are alive and active, and which enzymes and pathways function in soil microbial communities under different conditions.
Then we can begin to truly comprehend soil microbial communities from the microscopic to the global scale.Traumatic brain injury accounts for approximately 90% of brain injuries, and is associated with cognitive dysfunction and long-term disability. As a result of domestic incidents, military combat, traffic accidents and sports, TBI can compromise broad aspects of neuronal function. Patients often experience problems in the domains of learning, memory and affective functions that can profoundly influence quality of life. Existing therapeutic strategies for TBI have not been successful in counteracting the heterogeneous TBI pathology nor improving the quality of life of patients. Hence, identifying interventions with broad applicability seems necessary for effective management of TBI. Dietary polyphenols have significant positive effects on brain health via protecting neurons against injury and enhancing neuronal function. Evidence supports the neuromodulatory effects of flavonoid-rich blueberry, particularly in promotion of brain plasticity, and counteracting behavioural deficits. In the United States, demand for blueberries has increased, with 2017 fresh per capita consumption of 1.79 pounds/person. Several reports indicate that blueberry dietary supplementation improves memory, learning and general cognitive function, and protects against neuronal injury associated with stroke.
Moreover, it has been shown that blueberries possess potent antioxidant capacity through their ability to reduce free radical formation or upregulating endogenous antioxidant defenses. These studies suggest that blueberry supplementation can have the potential to be used to overcome the broad pathology of TBI. Given the lack of information about the effects of blueberry intake immediately after TBI, we have performed studies to assess the effects of blueberry extracts during the acute phase of TBI. Evidence suggests that TBI is characterized by dysfunction in synaptic plasticity, elevated levels of free radicals, plasma membrane dysfunction, which can contribute to the behavioural dysfunction. Oxidative stress is part of the pathology of TBI and compromises neuronal function. In particular, excessive free radical formation leads to accumulation of lipid oxidation by-products such as 4-hydroxynonenal with subsequent impairments in plasma membrane fluidity, receptor signaling across the membrane to deteriorate synaptic plasticity and reduce neuronal excitability. Deficiencies in brain derived neurotrophic factor reduce the brain plasticity necessary to cope with the effects of TBI. BDNF activates cAMP-responsive elementbinding protein , a multifaceted transcriptional regulator involved in synaptic plasticity essential for learning and memory. BDNF is known to bind to TrkB receptors, leading to activation of Ca2+/calmodulin-dependent protein kinase II , required for synaptic processes involved in behaviour. Several observations indicate that the flavonoids exert action through modulation of signaling pathways to promote synaptic and neuronal function.
Accordingly, in the current study, we investigated whether blueberry supplementation would counteract TBI pathology by involving BDNF related pathways involved in synaptic plasticity and oxidative stress to influence cognitive behaviours. Freeze-dried high bush whole blueberry fruit powder . This blend contained bio-active phytocompounds , and other macro- and micronutrients . Diet supplemented with 5% w/w BB was mixed with pulverized standard rodent chow . 1.6% fructose, 1.45% glucose and 0.0009% vitamin C were mixed with the standard rodent chow to match the levels of sugars in the BB supplemented diet and used as the rodent control diet . Sprague–Dawley male rats were purchased from Charles River Laboratories at 10 weeks of age and were acclimatized for vivarium 1 week prior to commencement of experimental procedures. Rats were housed in environmentally controlled conditions with 12-h light/dark cycle in a controlled room with free access to food and water. All procedures were approved by the University of California at Los Angeles Chancellor’s Animal Research Committee and were conducted with adherence to the guidelines set out by the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. After acclimatization, rats underwent fluid percussion injury or sham surgery and were pair housed to a specific diet group with either regular diet or blueberry supplemented diet for 2 weeks immediately. The BB dose was chosen based on previous in vivo studies which demonstrated that administration of blueberry offsets oxidative stress and reverses cognitive impairment. The groups were: Sham-RD as control group, TBI-RD, and TBI-BB. The rats were subjected to fluid percussion injury or sham surgery. Rats were subjected to learning on Barnes maze at post-injury day 14 for 5 days, and after a 2-day interval, memory was assessed at PID 21. Rats were tested for anxiety-like behaviour on elevated plus maze on PID 22 . All behavioural assessments were conducted between 9:00 and 13:00 hours. Rats were provided with diets prepared daily and fed ad libitum in powder form. To determine the voluntary food intake , food was weighed daily to measure consumption in each cage. Since the rats were pair-housed, food intake was divided by two to yield an approximate intake/rat.We employed our standard lateral fluid percussion injury protocol as described earlier. Briefly, 3% isoflurane was provided in a chamber , and then maintained with 2–2.5% isoflurane via nose cone while rats were in a stereotaxic frame. Body temperature was controlled by a heating pad. Under aseptic surgical conditions, a midline skin incision was made to expose the skull. Using a high-speed drill ,square planter pots craniotomy was made 3.0 mm posterior to bregma and 6.0 mm lateral of midline to expose the intact dura. A hollow plastic injury cap was placed over the craniotomy, secured with dental acrylic cement and was later filled with 0.9% saline. When the dental cement hardened, the anesthesia was discontinued and the rat was attached to the FPI device via the head cap. At the first response of hind-limb withdrawal to a paw pinch, rats received a moderate fluid percussion pulse . Upon resumption of spontaneous breathing the head cap was removed and the skin was sutured. Neomycin was applied on the suture and the rats were placed in a heated recovery chamber to be fully ambulatory before being returned to their cages. The sham animals were prepared using the identical surgically procedure but without the fluid pulse. Barnes maze testing was performed 2 weeks after experimental TBI with two trials per day with a 5-min test period . For learning assessment, rats were given two trials per day for five consecutive days at approximately the same time every day. Subsequently, memory retention was assessed at post-injury day 21. Latency to finding the escape hole and search strategies were analyzed for each trial.
Three search strategies were identified using following categorization: spatial, peripheral, and random using data recorded with AnyMaze software. EPM test was performed to assess anxiety-like behavior 2 weeks after experimental TBI with two trials as described in the Supplemental Information. Specifically, rats were individually placed in the closed arm of the EPM apparatus and permitted free exploration for 5 min during which their movements were camera recorded. The behaviors scored were time spent and number of entries into the open arm using automated video tracking system. EPM testing was conducted after Barnes maze memory test. Upon completion of the experiment, hippocampal tissues were harvested frozen in dry ice, and stored at –80 °C until use for immunoblotting. The left side hippocampus were homogenized in a lysis buffer containing 20 mM Tris–HCl , 137 mM NaCl, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonylfluoride , 10 μg/ml aprotinin, 0.1 mM benzethonium chloride, 0.5 mM sodium vanadate. Total protein was then determined using a BCA Protein Assay kit , using bovine serum albumin as standard. Equal amounts of protein were separated by sodiumdocecylsulphate-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes . Membranes were probed with anti-actin or anti-BDNF, anti-pCREB, anti-CREB, anti-CaMKII, anti-4- hydroxynonenal followed by secondary antibody . Immunoreactive proteins were visualized using enhanced chemiluminescence reagents . Band intensities were quantified using Image J32 Software. β-actin was used as an internal control for normalization western blot such that data were standardized according to β-actin values. Blots for each experimental group were normalized to ShamRD values within the same gel. Protein data are expressed as mean ± standard error of the mean . Body weight data expressed as mean ± standard deviation. Statistical analysis was performed by software GraphPad Prism 7.04. A level of 5% probability was considered as statistically significant. The Barnes maze learning data analysis were analyzed by repeated measures analysis of variance . Protein results are expressed as percentage of Sham-RD group.In the present study, we found that BB supplementation can attenuate important aspects of the acute TBI pathology. We report that BB supplementation immediately following TBI mitigates behavioural deficits in spatial learning and memory. BB supplementation counteracted the effects of TBI on proteins associated with the action of BDNF on plasticity and behaviour. In addition, BB supplementation counteracted the increase of the end product of lipid peroxidation, 4-HNE. The results showing that markers of neuronal plasticity and lipid peroxidation change in proportion to memory performance suggest a possible association between these molecular parameters and behaviour. Taken together, the present findings emphasize the beneficial effects of BB supplementation in fostering brain plasticity in the TBI pathology. In agreement with previous reports, we found that TBI impairs spatial learning as evidenced by an increase in latency in the Barnes maze, while BB supplementation decreased latency time to find the escape hole at each training day. We assessed the use of spatial learning strategies in our paradigm to provide a complementary measure of cognitive function less dependent on motor behaviour. Interestingly, we found that BB supplementation appeared to counteract a lost capacity of TBI rats to employ spatial leaning cues. This information together with results of the shorter latencies strongly suggest that BB supplementation protects TBI animals from a loss in spatial learning performance. In this regard, recent functional neuroimaging study in humans has established a connection between BB intake and cognitive function. Further, in the elevated plus maze test, rats exposed to TBI showed a tendency to reduce time spent in the open arms, which encompasses with clinical reports that psychiatric disorders are often observed in TBI patients. TBI-induced behavioural deficits probably stems from the impairments in BDNF-TrkB signaling that has been implicated in various cognitive and affective disorders. We cannot ascertain the cellular identity of the reported protein alterations. Although neuronal cells are the primary locus for learning and memory processing, non-neuronal cell types such as astrocytes and microglia can also contribute to these alterations. Moreover, it known that astrocytes and microglia provide support to synaptic transmission that is fundamental for neuronal function involved in cognitive processing.In the present investigation, we also found that TBI significantly reduced levels of hippocampal BDNF, and that BB dietary supplementation normalized these the levels. Previous report indicated that deficiencies in BDNF signaling is associated with impairments in cognition. Alternatively, cognition is strongly reliant on long-term potentiation and hippocampal BDNF, and the interaction between BDNF and its tyrosine kinase receptor is required for induction of LTP. Previously we have shown the protective effects of BDNF on the TBI pathology. Presently, our findings show that BB supplementation counteracted the BDNF reduction induced by TBI, paralleling improvements in cognitive function. It is well established that BDNF regulates synaptic plasticity and learning through interaction with the transcription factor CREB. Interestingly, our results also showed that BB supplementation normalized levels of CREB in TBI animals, and that these changes were proportional to changes in BDNF levels.