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Abstract Background Micronutrients have been associated with disease severity and poorer clinical outcomes in patients with COVID-19. However, there is a paucity of studies examining if the relationship with micronutrient status and clinical outcomes is independent of recognised prognostic factors, specifically frailty and the systemic inflammatory response (SIR). The aim of the present study was to examine the relationship between micronutrient status, frailty, systemic inflammation, and clinical outcomes in patients admitted with COVID-19. Methods Retrospective analysis of prospectively collected data was performed on patients with confirmed COVID-19, admitted to hospital between the 1st April 2020–6th July 2020. Clinicopathological characteristics, frailty assessment, biochemical and micronutrient laboratory results were recorded. Frailty status was determined using the Clinical Frailty scale. SIR was determined using serum CRP. Clinical outcomes of interest were oxygen requirement, ITU admission and 30-day mortality. Categorical variables were analysed using chi-square test and binary logistics regression analysis. Continuous variables were analysed using the Mann–Whitney U or Kruskal Wallis tests. Results 281 patients were included. 55% (n = 155) were aged ≥ 70 years and 39% (n = 109) were male. 49% (n = 138) of patients were frail (CFS > 3). 86% (n = 242) of patients had a serum CRP > 10 mg/L. On univariate analysis, frailty was significantly associated with thirty-day mortality (p

Background: An irreproducibility crisis currently afflicts a wide range of scientific disciplines, including public health and biomedical science. A study was undertaken to assess the reliability of a meta-analysis examining whether air quality components (carbon monoxide, particulate matter 10 µm and 2.5 µm (PM10 and PM2.5), sulfur dioxide, nitrogen dioxide and ozone) are risk factors for asthma exacerbation. Methods: The number of statistical tests and models were counted in 17 randomly selected base papers from 87 used in the meta-analysis. Confidence intervals from all 87 base papers were converted to p-values. p-value plots for each air component were constructed to evaluate the effect heterogeneity of the p-values. Results: The number of statistical tests possible in the 17 selected base papers was large, median = 15,360 (interquartile range = 1536–40,960), in comparison to results presented. Each p-value plot showed a two-component mixture with small p-values < 0.001 while other p-values appeared random (p-values > 0.05). Given potentially large numbers of statistical tests conducted in the 17 selected base papers, p-hacking cannot be ruled out as explanations for small p-values. Conclusions: Our interpretation of the meta-analysis is that random p-values indicating null associations are more plausible and the meta-analysis is unlikely to replicate in the absence of bias.

Background & AimsDiligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids.MethodsMouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors.ResultsIn contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures.ConclusionOur study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

Pancreatic ductal adenocarcinoma (PDAC) has traditionally been thought of as an immunologically quiescent tumor type presumably because of a relatively low tumor mutational burden (TMB) and poor responses to checkpoint blockade therapy. However, many PDAC tumors exhibit T cell inflamed phenotypes. The presence of tertiary lymphoid structures (TLS) has recently been shown to be predictive of checkpoint blockade response in melanomas and sarcomas, and are prognostic for survival in PDAC. In order to more comprehensively understand tumor immunity in PDAC patients with TLS, we performed RNA-seq, single and multiplex IHC, flow cytometry and predictive genomic analysis on treatment naïve, PDAC surgical specimens. Forty-six percent of tumors contained distinct T and B cell aggregates reflective of “early-stage TLS” (ES-TLS), which correlated with longer overall and progression-free survival. These tumors had greater CD8+ T cell infiltration but were not defined by previously published TLS gene-expression signatures. ES-TLS+ tumors were enriched for IgG1 class-switched memory B cells and memory CD4+ T cells, suggesting durable immunological memory persisted in these patients. We also observed the presence of active germinal centers (mature-TLS) in 31% of tumors with lymphocyte clusters, whose patients had long-term survival (median 56 months). M-TLS-positive tumors had equivalent overall T cell infiltration to ES-TLS, but were enriched for activated CD4+ memory cells, naive B cells and NK cells. Finally, using a TCGA-PDAC dataset, ES-TLS+ tumors harbored a decreased TMB, but M-TLS with germinal centers expressed significantly more MHCI-restricted neoantigens as determined by an in silico neoantigen prediction method. Interestingly, M-TLS+ tumors also had evidence of increased rates of B cell somatic hypermutation, suggesting that germinal centers form in the presence of high-quality tumor neoantigens leading to increased humoral immunity that confers improved survival for PDAC patients. AbbreviationsTLS: tertiary lymphoid structures; GC: germinal center(s); PDAC: pancreatic ductal adenocarcinoma; RNA-seq: RNA sequencing; BCRseq: B cell receptor sequencing; HEV: high endothelial venule; PNAd: peripheral node addressin; TMB: tumor mutational burden; TCGA: the cancer genome atlas; PAAD: pancreatic adenocarcinoma; FFPE: formalin fixed paraffin embedded; TIME: tumor immune microenvironment.

This is the Letter from the Editor.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes.

Abstract In the western United States, fire has become a significant concern in the management of big sagebrush (Artemisia tridentata Nutt.) ecosystems. This is due to large‐scale increases in cover of the fire‐prone invasive annual cheatgrass (Bromus tectorum L.) and, concurrently, concerns about declining quantity and quality of habitat for Greater Sage‐grouse (Centrocercus urophasianus). The prevailing paradigm is that fire results in a loss of sage‐grouse habitat on timescales relevant to conservation planning (i.e., 1–20 yr), since sagebrush cover can take many more years to recover post‐fire. However, fire can have effects that improve sage‐grouse habitat, including stimulating perennial grass and forb production. The conditions under which fire results in the permanent loss or enhancement of sage‐grouse habitat are not well understood. We used long‐term data from the Utah Division of Wildlife Resources Range Trend Project to assess short‐term (1–4 yr post‐treatment) and long‐term (6–10 yr post‐treatment) effects of fire on vegetation cover at 16 sites relative to sage‐grouse habitat vegetation guidelines. Sagebrush cover remained low post‐fire at sites considered historically unsuitable for sage‐grouse (10%) pre‐fire sagebrush cover, sagebrush cover decreased to 10% cover. Post‐fire sagebrush cover was positively related to elevation. Across all sites, perennial grasses and forbs increased in cover to approximately meet the habitat vegetation guidelines for sage‐grouse. Cheatgrass cover did not change in response to fire, and increased perennial grass cover appears to have played an important role in suppressing cheatgrass. Our results indicate that, while fire poses a potential risk for sage‐grouse habitat loss and degradation, burned sites do not necessarily need to be considered permanently altered, especially if they are located at higher elevation, have high sagebrush cover pre‐fire, and are reseeded with perennial grasses and forbs post‐fire. However, our results confirm that fire at more degraded sites, for example, those with

The Berryman Institute (BI) is a national organization based in the Department of Wildland Resources at Utah State University. We are dedicated to improving human–wildlife relationships and resolving human–wildlife conflicts through teaching, research, and extension.

The ability to restrict low molecular weight compounds to the gastrointestinal (GI) tract may enable an enhanced therapeutic index for molecular targets known to be associated with systemic toxicity. Using a triazolopyrazine CSF1R inhibitor scaffold, a broad range of prodrugs were synthesized and evaluated for enhanced delivery to the colon in mice. Subsequently, the preferred cyclodextrin prodrug moiety was appended to a number of CSF1R inhibitory active parent molecules, enabling GI-restricted delivery. Evaluation of a cyclodextrin prodrug in a dextran sodium sulfate (DSS)-induced mouse colitis model resulted in enhanced GI tissue levels of active parent. At a dose where no significant depletion of systemic monocytes were detected, the degree of pharmacodynamic effect-measured as reduction in macrophages in the colon-was inferior to that observed with a systemically available positive control. This suggests that a suitable therapeutic index cannot be achieved with CSF1R inhibition by using GI-restricted delivery in mice. However, these efforts provide a comprehensive frame-work in which to pursue additional gut-restricted delivery strategies for future GI targets.

Human–Wildlife Interactions is seeking contributed original science and/ or synthesis and review papers for a special section on wild horse and burro management to be published in March/April 2018. The Bureau of Land Management (BLM) created the Wild Horse and Burro Program to implement the Wild-Free Roaming Horses and Burros Act, passed by Congress in 1971. Broadly, the law stipulates that the BLM and the U.S. Forest Service have the responsibility to manage and protect herds in their respective jurisdictions within areas where wild horses and burros were found roaming in 1971. Bureau of Land Management rangelands can only support around 27,000 horses and burros, yet nearly 73,000 inhabit these ranges and >45,000 are held in off - range facilities, which cost about $50 million each year. By next year, the on range population could grow another 15–20%. The goal of the special section synthesizes the science, management, and policy regarding implementation of the 1971 Wild Horse and Burro Act and the management of wild horses and burros worldwide.

The Berryman Institute (BI) is a national organization based in the Department of Wildland Resources at Utah State University. We are dedicated to improving human– wildlife relationships and resolving human– wildlife conflicts through teaching, research, and extension.

The U.S. Fish and Wildlife Service (USFWS) designated greater sage-grouse (Centrocercus urophasianus) as a candidate species to receive protection under the Endangered Species Act in 2010. Several states in the western United States have developed management plans to mitigate the listing factors identified by the USFWS. However, sage-grouse populations inhabit sagebrush (Artemisia spp.) ecosystems that may transcend multiple state boundaries. If sage-grouse inter-seasonal movements encompass habitats in multiple states and if state-centric wildlife management strategies differ, species conservation may be further complicated. Additionally, if these populations are located in peripheral state boundary areas, they may also receive less management focus both because of their remoteness and state agency emphasis on interior populations. The Grouse Creek sage-grouse population that inhabits extreme northwestern Utah where the state borders eastern Nevada and southern Idaho exemplifies this situation. We monitored 50 radio-collared sage-grouse from 2005 to 2006 to document inter-seasonal movements relative to sex and age and state boundaries. Radio-collared sage-grouse migrated an average of 13.1 km from breeding (range = 0.2 to 69.3, SE = 14.4) to summer range, 22.6 km from summer (range = 0.2 to 46.1, SE = 12.6) to winter range, and 25.4 km from winter (range = 1.1 to 37.2, SE = 13.4) to return to spring range. Ten radio-marked birds (20%) used seasonal habitats in Idaho, Nevada, and Utah. Males were more likely to engage in long-distance movements than females during the breeding season. We confirmed that within a geographically defined state population, individuals may exhibit diverse inter-seasonal migration strategies. Our results support the need for increased coordination among states that share occupied sagebrush habitats to develop interstate sage-grouse conservation plans.

Competency, its nature, its acquisition and its maintenance, is emerging as the pivotal dimension of human development for employability, corporate competitive advantage and national economic success. It is, however, a concept still fogged by misunderstanding and debate. The authors of this article attempt to clear the fog by providing a succinct conceptual framework which integrates the contributions of many theorists and accommodates the use of the term in a variety of contexts. In addition, they propose that competencies are more than the essence of a national qualifications framework, or of corporate training programmes, as they are intimately linked to strategy with all its implications. Competency, therefore, is a central issue for managers seeking competitive advantage.

Dual tasks requiring sustained visual attention and upright stance are common, yet their impact on standing balance is not well understood. We investigated the role of visual attention in the maintenance of postural control, using the multiple-object tracking (MOT) task. Healthy young adults ( n = 12) performed the MOT task at three object movement speeds while seated or standing. MOT performance was assessed using tracking capacity ( k ). Metrics calculated to assess mediolateral (ML) and anterior-posterior (AP) postural control included: maximum difference between CoM and CoP position (CoM-CoP Max), root mean square distance for center of pressure and center of mass position (CoP and CoM RMS distance), and correlation between CoM and CoP time series signals (CoM/CoP correlation). As predicted, k decreased significantly as object movement speed increased for both standing and seated conditions. Object movement speed also significantly affected AP CoM-CoP Max in seated conditions ( p = .021) and AP CoM/CoP correlation for standing conditions ( p = .002). The results demonstrate utility of the MOT task in understanding the role of visual attention in postural control, even though healthy young adults were able to compensate for the addition of a sustained visual attention task, with minimal deficits to postural control.

Multiple-object tracking (MOT) involves keeping track of the positions of multiple independent target items as they move among distractors. According to Pylyshyn (Cognition, 80, 127-158, 2001), the item individuation mechanism used in MOT is also used in visually guided touch. To test this, we compared single-task MOT (MOT alone) with dual-task MOT (MOT while touching items that changed colour), looking for interference: cases where single-task performance was worse than dual-task. Touching items that changed colour interfered with MOT, but effects varied depending on whether the item touched was a target or distractor in MOT. Touching distractors always reduced MOT performance more than touching targets. Touching targets during MOT did not interfere when there was only a single target to track but interfered more once there were two or more targets. We also measured interference based on latencies to touch items that changed colour, comparing single and dual-task conditions (touch alone, touch + track). MOT interfered with touch, increasing RT to touch items that changed colour, with latencies significantly higher when those items were distractors rather than targets. Overall, there was general interference (differences between single and dual-task performance), as might be expected if coordinating the two tasks required a common limited resource such as general attention or working memory. However, there was also differential interference that varied based on whether the touched item was a target or distractor in MOT. This differential interference suggests the specific mechanisms used in MOT may also play a role in visually guided touch.

This study evaluated the effects of 4 different weekly stretching protocols on the rate of gain and decline in hamstring flexibility over an 8-week period, across sex. Using a randomized single-blind design, 53 healthy subjects aged 18-46 years were assigned to 1 of 4 stretching protocols or a control group. The stretching protocols consisted of either daily or 3 times per week stretching and performed once or twice each day. These protocols differed in terms of frequency and total weekly stretching time. All the subjects stretched their hamstring muscles for 4 weeks and were measured weekly for their hip range of motion (ROM). Stretching ceased the final 4 weeks as the weekly measurements continued. The results revealed no significant differences in the rate of gain or the rate of loss between the different stretching protocols (2-way analysis of variance, F = 2.60, p > 0.05). All the stretching groups gained in hip ROM from pre to week 4 (F = 269.24, p < 0.001). After cessation, the rate of loss was similar for all the 4 stretching groups (F = 102.86, p < 0.001); all the groups retained significant gains at the end of the study (p < 0.001). The control group did not change over time. Those who stretched at least 6 times per week gained more than those who stretched 3 times per week (24 and 16.8%, respectively, F = 5.20, p < 0.05). Subject sex did not influence ROM changes (p > 0.05). Stretching appears to be equally effective, whether performed daily or 3 times per week, provided individuals stretch at least 2 times each day. Moreover, although women are more flexible than men are, there was no sex difference in terms of stretching response.

Despite recent attention in the literature, anterior cruciate ligament (ACL) injury mechanisms are controversial and incidence rates remain high. One explanation is limited data on in vivo ACL strain during high-risk, dynamic movements. The objective of this study was to quantify ACL strain during jump landing. Marker-based motion analysis techniques were integrated with fluoroscopic and magnetic resonance (MR) imaging techniques to measure dynamic ACL strain non-invasively. First, eight subjects' knees were imaged using MR. From these images, the cortical bone and ACL attachment sites of the tibia and femur were outlined to create 3D models. Subjects underwent motion analysis while jump landing using reflective markers placed directly on the skin around the knee. Next, biplanar fluoroscopic images were taken with the markers in place so that the relative positions of each marker to the underlying bone could be quantified. Numerical optimization allowed jumping kinematics to be superimposed on the knee model, thus reproducing the dynamic in vivo joint motion. ACL length, knee flexion, and ground reaction force were measured. During jump landing, average ACL strain peaked 55±14 ms (mean and 95% confidence interval) prior to ground impact, when knee flexion angles were lowest. The peak ACL strain, measured relative to its length during MR imaging, was 12±7%. The observed trends were consistent with previously described neuromuscular patterns. Unrestricted by field of view or low sampling rate, this novel approach provides a means to measure kinematic patterns that elevate ACL strains and that provide new insights into ACL injury mechanisms., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: The influence of superficial precooling on range of motion (ROM) as part of a stretching program has not been extensively studied. It is not clear if the analgesic effect can benefit a stretching program., Hypotheses: Superficial precooling will result in greater gains in ROM as part of a stretching program, compared with stretching without a precooling intervention. Superficial precooling will also result in greater retention in ROM gains following cessation of stretching, compared with stretching without a precooling intervention., Study Design: Prospective randomized single-blind test-retest design., Methods: Twenty-nine participants were randomly assigned to 1 of 2 static stretching protocols: a standard protocol (n, 14; age, 24.6 ± 5.4 years) or a precool protocol (n, 15; age, 25.1 ± 7.3 years). These samples allowed for 80% power for statistical significance testing. Both groups performed static hamstring stretching daily for 4 weeks. The precool group applied ice to the hamstring for 10 minutes before stretching. Both groups stretched for 4 weeks and then stopped stretching for the last 4 weeks. Hip ROM measures were obtained each week for 8 weeks., Results: For the standard group, mean hip ROM increased from 71.4° ± 18.5° to 90.6° ± 20.5° and for the precool group, 71.5° ± 22.3° to 91.8° ± 20.9°. For the standard group, mean hip ROM decreased from 90.6° ± 20.5° to 83.9° ± 20.3° and for the precool group, 91.8° ± 20.9 to 85.0° ± 19.4°. There were no differences between groups at any time in the study (P > .05)., Conclusions: Precooling had no beneficial effects on ROM or on retention of ROM., Clinical Relevance: Cold application, before stretching, does not provide any benefit to a stretching program.

Bursts of activity in the surface electromyogram (EMG) during a sustained contraction have been interpreted as corresponding to the transient recruitment of motor units, but this association has never been confirmed. The current study compared the timing of trains of action potentials discharged by single motor units during a sustained contraction with the bursts of activity detected in the surface EMG signal. The 20 motor units from 6 subjects [recruitment threshold, 35.3 +/- 11.3% maximal voluntary contraction (MVC) force] that were detected with fine wire electrodes discharged 2-9 trains of action potentials (7.2 +/- 5.6 s in duration) when recruited during a contraction that was sustained at a force below its recruitment threshold (target force, 25.4 +/- 10.6% MVC force). High-pass filtering the bipolar surface EMG signal improved its correlation with the single motor unit signal. An algorithm applied to the surface EMG was able to detect 75% of the trains of motor unit action potentials. The results indicate that bursts of activity in the surface EMG during a constant-force contraction correspond to the transient recruitment of higher-threshold motor units in healthy individuals, and these results could assist in the diagnosis and design of treatment in individuals who demonstrate deficits in motor unit activation.

A technique of centrifuging pea epicotyl sections which extracts water-soluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests for protein, hexose, pentose, and malate dehydrogenase indicate that significant damage to the cells occurs above 3,000g. Below this force, there is little damage, as evidenced by the similar growth rates of centrifuged and noncentrifuged sections. Centrifugation at 1,000g extracts polysaccharides containing rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. An increase in xylose and glucose, presumably xyloglucan, is induced by treating sections with indoleacetic acid. Much of the alcohol-insoluble, water-soluble polysaccharide within the wall is extractable by centrifugation, since nearly as much arabinose and xylose are extractable by centrifugation as by homogenization. The utility of this method for the study of cell wall metabolism is discussed.

A protein identifiable as calmodulin has been isolated from oat (Avena sativa, var Garry) tissues. This protein is relatively heat stable, binds to hydrophobic gels, and phenothiazines in a calcium-dependent fashion, and binds to antibody to rat testes calmodulin. Based on its migration on sodium dodecyl sulfate-polyacrylamide gels, ultraviolet absorption spectrum, and amino acid composition, oat calmodulin is essentially identical to calmodulin isolated from other higher plants. Radioimmunoassays indicate that calmodulin is associated with isolated oat protoplasts, mitochondria, etioplasts, and nuclei and also appears to be a component of oat cell wall fractions.

Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv ;Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10(10) cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10(10) bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10(10) bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.To test the hypothesis that this reduced synthesis of exopolysaccharide by bacteroids is related to the low free O(2) concentration within nodules, strain RT2 was grown on l-arabinose/succinate/glutamate/nitrate medium both aerobically and anaerobically. Anaerobiosis caused a 92% reduction in total exopolysaccharide synthesis, with amounts averaging only 123 micrograms per 10(10) cells. Anaerobically cultured cells also failed to bind soybean lectin. These results suggest that the low free O(2) content of the nodules may be responsible for the reduced exopolysaccharide synthesis by the bacteroids.

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

The effect of ethylene on cell wall metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fresh weight of excised, 1-cm sections. Cell wall metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wall solution extracted with H(2)O are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose and galactose levels are not altered by ethylene until 8 hours of treatment, whereupon they decline in controls in air relative to ethylene treatment. When alcohol-insoluble polymers are fractionated into neutral and acidic polysaccharides, xylose and glucose predominate in the neutral fraction and arabinose and galactose in the acidic fraction. Ethylene depresses the levels of xylose and glucose in the neutral fraction and elevates arabinose and galactose in the acidic fraction. Ethylene treatment does not affect the level of uronic acids extracted with H(2)O; however, the level of hydroxyproline-rich proteins in this water-extracted cell wall solution is increased by ethylene. Extraction of sections with CaCl(2) results in an increase in the levels of neutral sugars particularly arabinose. Ethylene depresses the yield of arabinose in calcium-extracted solution relative to controls in air. Similarly, extraction with CaCl(2) increases the yield of extracted hydroxyproline in ethanol-insoluble polymers and ethylene depresses its level relative to controls. Metabolism of uronic acids and neutral sugars and growth in response to ethylene treatment contrast markedly with auxin-induced polysaccharide metabolism and growth. With auxin, sections increase mostly in length not radius, and this growth form is associated with an increase in the levels of xylose, glucose, and uronic acids. With ethylene, on the other hand, stem elongation is suppressed and expansion is promoted, and this growth pattern is associated with a decrease in xylose and glucose in the ethanol-insoluble polysaccharides.

The metabolism of polysaccharides by pea stem segments treated with and without auxin was investigated using a centrifugation technique for removing solution from the free space of the cell wall. Glucose is the predominant sugar in both the ethanol-soluble and ethanol-insoluble fractions of the cell wall solution extracted with water. In the water-soluble, ethanol-insoluble polysaccharides, arabinose, xylose, galactose, and glucose make up 9.5, 23.8, 23.9, and 39.9%, respectively, of the neutral sugars, while rhamnose, fucose, and mannose are present at concentrations between 0.5 and 2.0%.Auxin treatment enhances the levels of xylose and glucose in ethanol-insoluble polysaccharides relative to controls, and this difference can be detected within 30 minutes of auxin treatment. Cellulose-binding experiments show that the enhanced levels of xylose and glucose are in a polymer having the cellulose-binding properties of xyloglucan. (3)H-glucose labeling experiments confirm the auxin-enhanced metabolism of the xyloglucan fraction; however, increased labeling of arabinose is also observed in auxin-treated sections. Auxin treatment also causes a marked increase in the level of uronic acids centrifuged from pea internode sections. Thus, after 3 hours of incubation in indoleacetic acid, the level of uronic acids in the ethanol-insoluble polysaccharides which can be recovered by centrifugation is increased 2- to 3-fold over sections incubated in water. These auxin-enhanced changes in xylose, glucose, and uronic acids are correlated with enhanced rates of section growth.Incubation of excised pea internode sections in acidic buffers also enhances the rate of xyloglucan and polyuronide metabolism. This acid-enhanced metabolism of xyloglucan and polyuronide is inhibited by low temperature, suggesting that it is enzyme-mediated.Extraction of the cell wall solution with CaCl(2) increases the yield of all neutral sugars. Arabinose and mannose are increased 4- and 3-fold, respectively, and xylose and glucose by about 20%, while galactose levels are 40% higher in cell wall solution extracted with CaCl(2) than in that extracted with water. Although calcium increases the amount of neutral sugars extracted, it does not affect the auxin-induced changes in neutral sugars. Extraction of the cell wall solution with ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'tetraacetic acid enhances the yield of uronic acids and also increases the difference due to auxin treatment.

Auxin-induced elongation and cell wall polysaccharide metabolism were studied in excised hypocotyl sections of ponderosa pine (Pinus ponderosa) seedlings. Sections excised from hypocotyls of ponderosa pine elongate in response to the addition of auxin. The neutral sugar composition of the extracellular solution removed from hypocotyl sections by centrifugation was examined. In cell wall solution from freshly excised sections, glucose, galactose, xylose, and arabinose make up more than 90% of the neutral sugars, while rhamnose, fucose, and mannose are relatively minor components. The neutral sugar composition of the polysaccharides of the pine cell wall solution is both qualitatively and quantitatively similar to that of pea. Following auxin treatment of pine hypocotyls, the neutral sugar composition of the cell wall changes; glucose, xylose, rhamnose, and fucose increase by nearly 2-fold relative to controls in buffer without auxin. These changes in neutral sugars in response to auxin treatment are similar to those found in pea, with the exception that in pea, rhamnose levels decline in response to auxin treatment.

The capacity of excised internode sections of pea to grow and secrete protons in response to indoleacetic acid (IAA) and Ca(2+) and K(+) treatments was examined. By incubating unpeeled and unabraded sections in rapidly flowing solutions, it was shown that acidification of the external medium in the presence or absence of IAA is dependent on the presence of Ca(2+) and K(+). Similar results were obtained when unpeeled and unabraded sections were incubated in dishes with shaking. When peeled or abraded sections were incubated with shaking in IAA, H(+) release was also dependent on the presence of Ca(2+) and K(+). The release of H(+) from sections incubated in Ca(2+) and K(+) is not caused by displacement of H(+) from binding sites in the cell wall. Rather, the release of protons from sections is temperature dependent, and it is concluded that this is a metabolically linked process. Although Ca(2+) and K(+) are essential for the release of H(+) from isolated stem sections of peas, these cations do not influence elongation. Despite the large increase in proton release induced by Ca(2+) and K(+) either in the presence or absence of auxin, growth in the presence of these ions was never greater than it was in their absence. Furthermore, cations do not affect the neutral sugar or uronic acid composition of the solution which can be centrifuged from isolated sections. As is the case for growth, an increase in the neutral sugar and uronide composition of the cell wall solution is dependent only on IAA. It is concluded that IAA-induced growth of pea stem sections is independent of the secretion of protons.