Development of alfalfa strains with differential tolerance to aluminum toxicity

Summary Aluminum toxicity limits root growth in acid subsoils that are difficult to lime. An alternative to subsoil liming is the development of plants having greater tolerance to Al. Alfalfa (Medicago sativa L.) is considered an Al-susceptible species. Preliminary studies indicated that alfalfa cultivars differ in Al tolerance, but the extreme plant-to-plant variation within cultivars prevented the establishment of clearcut cultivar differences.Tolerant and susceptible plants were selected from each of six cultivars (DuPuits, Atlantic, Team, Buffalo, Grimm, and Sirsa 9) grown on an Al-toxic Bladen soil at pH 4.1 to 4.3. The tolerant selections were repotted and interpollinated to form one population of polycross seed. Susceptible selections were treated similarly to form a second population. These two populations, tolerant and susceptible, were subjected to an additional cycle of recurrent phenotypic selection for tolerance and susceptibility, respectively, to Al-toxic Bladen soil at pH 4.6.Plants from the population selected for tolerance to the acid Bladen soil were significantly higher in both root and top vigor on Al-toxic Tatum soil than plants from the population selected for susceptibility. The results indicated that Al tolerance is a heritable trait in these alfalfa populations and that recurrent selection can be used effectively to develop strains having differential tolerance to Al-toxic soils. The observation that only 2% of the plants from the tolerant population were in the most tolerant class suggests a good opportunity for more progress in selecting toward Al tolerance.     

Ion dependence of the tarsal sugar receptor of the blowfly Phormia regina.

The activity of the tarsal sugar receptor is greatly reduced following prolonged water exposure. The animal's behavior, which characteristícally reflects receptor input, also shows decreased acceptance of sucrose solutions following prolonged tarsal immersion in deionized water. Long exposure of the tarsi to Bodenstein's saline instead of water does not produce as large a decrement in the acceptance response as does water exposure.Recovery of the behavioral response occurs spontaneously after a few hours. The original response level can also be restored immediately if a moderate concentration (0.05 to 0.2 M) of KCl or NaCl is added to the sucrose stimulus. The effect of LiCl is ambiguous: it inhibits the normal sucrose response, thereby tending to mask any restorative effects. The electrophysiological data show that the cellular response level is also restored when Na⁺ or K⁺ ions are present in the stimulus.The above data are interpreted to mean that the effect of tarsal water exposure is to slowly leach out ions in the effective extracellular fluid surrounding the receptor membrane, thus lowering the membrane potential and deceasing the receptor potential upon stimulation. The fact that Na⁺ and K⁺ when supplied in the stimulating solution temporarily restore the original response level suggests that these extrinsically added ions can be used as current carrying ions to depolarize the cell. The data suggest that the sensillum contains three functional compartments interconnected by partial diffusion barriers: (1) a ‘receptor compartment’ (2) an axial cylinder which contains the dendrites and functions as the immediate extracellular ion source, and (3) a larger axial cylinder which serves as an ion reservoir.A method for statistically analyzing behavioral acceptance data is presented.     

T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.     

Intrathecal Morphine Use in Adolescent Idiopathic Scoliosis Surgery is Associated with Decreased Opioid Use and Decreased Length of Stay

BackgroundLength of stay (LOS) in the hospital following posterior spinal fusion (PSF) for adolescent idiopathic scoliosis (AIS) has decreased over the past decade due to well-defined postoperative clinical pathways, earlier mobilization, and improved pain control methods. Historically, liberal use of parenteral and oral opioids for pain control caused side effects, resulting in delayed discharge. Intraoperative intrathecal morphine (ITM) has been posited to reduce the need for postoperative opioids and to expedite the discharge process. This study examines the relationship between the use of ITM with average required postoperative opioid usage and with average LOS.MethodsThis IRB-approved retrospective cohort study examined 105 patients with AIS who received PSF with instrumentation split into two cohorts. One cohort underwent PSF via standard surgical protocol (n=40) while the other cohort received intraoperative ITM with the standard surgical protocol (n=65). Power analysis demonstrated a study power of 0.8. LOS and total postoperative opioid analgesic medication (morphine milligram equivalent, MME) data were collected. Age at surgery, gender, number of spinal levels fused, estimated intraoperative blood loss (EBL), preoperative Cobb angle, and any complications related to the use of ITM were also recorded. Continuous variables were analyzed with Student’s t-test and categorical variables were analyzed with chi-square independent-sample tests using SAS 9.4 (α = 0.05).ResultsPatients who were treated with ITM displayed shorter LOS (p<0.0001) and reduced postoperative analgesic requirement (p<0.0001). Patients who received ITM spent an average of 1.8 fewer midnights in the hospital and received an average of 221.2 MME less than patients who received standard protocol (57% decrease). There were no significant differences between the two groups for any other variable.ConclusionIntraoperative ITM is a simple and effective treatment for scoliosis surgeons to better control postoperative pain in patients, reduce the risk of dependency, and achieve earlier discharge from the hospital. Shortened LOS reduces the overall cost of care, benefitting patients, hospitals, and insurance companies. Based on the results of this study and several earlier studies, the authors recommended that scoliosis surgeons consider incorporating use of ITM into their standard operative protocols. Level of Evidence: IV     

Alleviation of Both Water and Nutrient Limitations is Necessary to Accelerate Ecological Restoration of Waste Rock Tips

Hard rock quarries are commonly located close to national parks and special areas of conservation and are generally regarded as visually intrusive. Consequently, restoration strategies that effectively accelerate natural plant regeneration processes are required. Slate waste tips present extreme conditions for plant establishment with multiple potential limiting factors (e.g., lack of organic matter, nutrients, and poor water retention). In this study, we investigated ecological strategies to accelerate natural regeneration at the largest slate quarry in Europe. A field experiment was conducted to assess ecosystem restoration using a contrasting set of native woody species. Treatments included amendments of waste tips with: polyacrylamide gel to increase water‐holding capacity; mineral fertilizer to increase nutrient supply; and two treatments that increased both (organic waste or boulder clay addition). Ecosystem recovery was evaluated through above‐ and below‐ground productivity (plant and microbial, respectively) and soil analyses. Neither increasing nutrient supply (with mineral fertilizer) nor water‐holding capacity (with polyacrylamide gel) was sufficient, alone, to improve plant establishment. However, both boulder clay and organic waste amendment significantly enhanced plant growth. There was a marked positive interaction in the effects on tree growth of the amendment with organic waste and boulder clay. Large interactions occurred between tree species and substrate amendments. The growth of N₂‐fixing species was strongly favored over non‐fixers where there was no addition of material increasing soil nitrogen supply, whereas the growth advantage of pioneer species over non‐pioneers was greatest with fertilizer, organic waste, or clay additions. Organic waste addition had the greatest positive impact on soil processes.     

Comparison of vascular smooth muscle cells from adult human,monkey and rabbit in primary culture and in subculture

Summary A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or dedifferentiated after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or dedifferentiation process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of dedifferentiated cells at all times.The advantages of differentiated rather than dedifferentiated smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilitiesHolder of a John Halliday Travelling Fellowship from the Life Insurance Medical Research Fund of Australia and New ZealandResearch Fellow with the National Heart Foundation of AustraliaSupported by the Deutsche Forschungsgemeinschaft