BackgroundPatient outcomes are influenced by many confounding factors peri-operatively, including the type of surgery, anaesthesia, transfusion, and immune competence. We have previously demonstrated (in-vitro) that compared to allogeneic blood transfusion (ABT), intraoperative cell salvage (ICS) improves immune competence. The peri-operative immune response is complex. Altered or impaired immune responses may predispose patients to develop adverse outcomes (i.e., post-operative wound infection, pneumonia, urinary tract infection etc.) Surgical patients may develop infection, even without the confirmed presence of a definite microbiological pathogen. With all these factors in mind it is important to consider changes in immune cell numbers (and sub-populations) and functional capacity during peri-operative transfusion. MethodsIn this TRIMICS-Cell (Transfusion Related Immune Modulation and Intraoperative Cell Salvage-Cell numbers) study (n = 17, October 2018-November 2019) we prioritized and analysed peri-operative changes in the number and proportions of immune cell populations and sub-populations (B cells (CD20(+)), NK (natural killer) cells (CD56(+)), monocytes (CD14(+)), T cells (total CD3(+) and sub-populations: T helper cells (CD4(+)), cytotoxic T cells (CD8(+)), effector T cells (CD4(+) CD127(+)), activated effector T cells (CD4(+) CD25(+) CD127(+)) and regulatory T cells (CD4(+) CD25(+) CD127(-))), plasmacytoid dendritic cells (pDC; Lineage(-), HLA-DR+, CD11c(-), CD123(+)), classical dendritic cell (cDC) (Lineage(-), HLA-DR+, CD11c(+)), and cDC activation (Lineage(-), HLA-DR+, CD11c(+)), co-stimulatory/adhesion molecules and pDC (CD9(+), CD38(+), CD80(+), CD83(+), CD86(+), CD123(+)). Firstly we analysed the whole cohort of study patients and secondly according to the relevant transfusion modality (i.e., three study groups: those who received no transfusion, received ICS only (ICS), or both ICS and allogeneic packed red blood cells (pRBC) (ICS & RBC)), during major orthopaedic surgery. ResultsFor the whole study cohort (all patients), changes in immune cell populations were significant: leucocytes and specifically neutrophils increased post-operatively, returning towards pre-operative numbers by 48h post-operatively (48h), and lymphocytes reduced post-operatively returning to pre-operative numbers by 48h. When considering transfusion modalities, there were no significant peri-operative changes in the no transfusion group for all immune cell populations studied (cell numbers and proportions (%)). Significant changes in cell population numbers (i.e., leucocytes, neutrophils and lymphocytes) were identified in both transfused groups (ICS and ICS & RBC). Considering all patients, changes in immune cell sub-populations (NK cells, monocytes, B cells, T cells and DCs) and functional characteristics (e.g., co-stimulation markers, adhesion, activation, and regulation) were significant peri-operatively and when considering transfusion modalities. Interestingly DC numbers and functional capacity were specifically altered following ICS compared to ICS & RBC and pDCs were relatively preserved post-operatively following ICS. ConclusionA transient peri-operative alteration with recovery towards pre-operative numbers by 48h post-surgery was demonstrated for many immune cell populations and sub-populations throughout. Immune cell sub-populations and functional characteristics were similar peri-operatively in those who received no transfusion but changed significantly following ICS and ICS & RBC. Interesting changes that require future study are a post-operative monocyte increase in the ICS & RBC group, changes in cDC considering transfusion modalities, and possibly preserved pDC numbers post-operatively following ICS. Future studies to assess changes in immune cell sub-populations, especially during peri-operative transfusion, while considering post-operative adverse outcomes, is recommended.

SUMMARYThroughout life, skeletal muscle, the arbiter of voluntary movements, is maintained by a population of skeletal muscle-dedicated stem cells, called muscle satellite cells (MuSCs). Similar to other adult stem cells, the function of MuSCs is tightly coordinated by the cellular and acellular components of their microenvironment, or the niche. While the processes that control the coupling of neurotransmission and muscle contraction have been well characterized, little is known on the reciprocal crosstalk between neural cells and MuSCs within the muscle microenvironment. Here, we report that mild peripheral nerve injury enhances MuSC myogenic function and muscle regeneration by synergistically augmenting MuSC mitochondrial bioenergetics and upregulating anabolic protein synthesis pathways. We also demonstrate that chronic disruption or degeneration of neuromuscular synapses, such as in muscular dystrophy and biological aging, abolishes MuSC and motor neuron interactions, causing significant deficits in muscle regeneration following injury. These results underscore the importance of neuromuscular junction and neural network as an essential niche of MuSCs. Determining the significance of MuSC-nerve interactions and their functional outcomes, as well as the possibility of modulating these connections, have important implications for our understanding of neuromuscular disease pathology and development of therapeutic interventions.HighlightsMild peripheral nerve injury increases muscle stem cell bioavailability of healthy muscle.Nerve perturbation stimulates myogenesis by enhancing protein synthesis and mitochondrial metabolism in young, healthy muscle.Synergistic crosstalk within neuromuscular niche boosts muscle regeneration in young, healthy muscle.Positive influences from the neural network on muscle stem cells are abolished in pathological denervation manifested in dystrophic and aging muscle.

ABO compatible allogeneic blood transfusion (ABT) is associated with transfusion-related immune modulation (TRIM) and poor patient outcomes including perioperative infection and myocardial infarction. Intraoperative cell salvage (ICS) enables collection, processing and reinfusion of autologous blood lost during surgery. We investigated whether immune suppression associated with ABT could be reduced by using ICS. Suitable orthopaedic patients were consented and recruited to the study (n=20, HREC/17/QRBW/685). 10mL anticoagulated blood (EDTA) was collected from the patient (pre-operatively) and from the ICS blood product. An established whole blood culture model was used to assess changes to monocyte and myeloid dendritic cell (mDC) inflammatory responses (intracellular cytokine staining for IL-6, IL-8, IL-10, IL-12, IL-1α, TNF-α, MIP-1α, MIP-1β, MCP-1, IP-10) following exposure of the patient’s pre-operative blood to ABO compatible allogenic blood (Blood Service, Kelvin Grove) or their ICS blood (P<0.05 ANOVA). Exposure to ABT significantly suppressed the inflammatory response of both mDC (IL-10, IL-12, IL-1α, IL-8, TNF-a, MIP-α, MIP-1β) and monocytes (IL-10, IL-6, IL-12, IL-1α, IL-8, IL-10, IL-12, TNF-α and MIP-1α). Compared to ABT, exposure to the patients’ own (ICS) blood improved mDC IL-8, IL-10, IL-12, TNF-α and MIP-1α production and monocyte IL-10 and MIP-1β production. We provide in vitro evidence that ICS may reduce ABT associated adverse outcomes associated with improved inflammatory response and immune competence post-transfusion.

Lateral roots originate from initial cells deep within the main root and must emerge through several overlying layers. Lateral root emergence requires the outgrowth of the new primordium (LRP) to coincide with the timely separation of overlying root cells, a developmental program coordinated by the hormone auxin. Here, we report that in Arabidopsis thaliana roots, auxin controls the spatiotemporal expression of the plasmodesmal regulator PDLP5 in cells overlying LRP, creating a negative feedback loop. PDLP5, which functions to restrict the cell-to-cell movement of signals via plasmodesmata, is induced by auxin in cells overlying LRP in a progressive manner. PDLP5 localizes to plasmodesmata in these cells and negatively impacts organ emergence as well as overall root branching. We present a model, incorporating the spatiotemporal expression of PDLP5 in LRP-overlying cells into known auxin-regulated LRP-overlying cell separation pathways, and speculate how PDLP5 may function to negatively regulate the lateral root emergence process.

One risk of allogeneic blood transfusion is the development of alloantibodies against red blood cell (RBC) antigens not found on the recipient’s RBC. The monocyte monolayer assay (MMA) is an in vitro model which has been used to determine the clinical significance of alloantibodies and predict post-transfusion RBC survival. We aimed to develop a semi-automated procedure to reduce assay time and facilitate improved patient management. PBMCs isolated using Sepmate tubes (STEMCELL Technologies) were added to Lab-Tek 8-well chamber slides (37°C, 1 hr, CO2). Antigen matched RBC were sensitised with plasma with known anti-RBC antibodies (anti-D n=3, anti-Yta n=4) (37°C, 1 hr, CO2). A modified Wright-Giemsa staining protocol was performed (Aerospray Hematology Pro 2 stainer, ELITechGroup) and the proportion of bound and/or phagocytosed RBC was determined via microscopy (>5% considered clinically significant). The introduction of PBMC isolation using sepmate tubes and automated cell staining reduced technical requirements, improved assay reproducibility and reduced assay time by >2 hours. For the alloantibodies tested, 67% of the anti-D and none of the anti-Yta were clinically significant. We developed a MMA with improved reproducibility and reduced turnaround time. This assay will improve blood transfusion safety and facilitate better patient management by reducing the risk of incompatible blood transfusion.

Background: Pediatric pneumonia causing bacteremia is a severe condition and the appropriate antibiotic treatment is the mainstay to save their life. Aims: To describe bacterial characteristics of bacteremia in pediatric patients with pneumonia Methods: A retrospective study was conducted at Children hospital no 1, Vietnam from January 2016 to December 2017. All subjects with age ≤ 16 years old had clinical and radiological manifestations consistent to pneumonia and the positive result of blood culture were included. Results: We found 37 of eligible pediatric patients in which 16 patients were excluded because of the blood culture positive for coagulase-negative Staphylococcus. This study showed 54.1% patients with history of underlying condition, 86.5% with duration of symptom ≤ 7 days, and 16.2% with septic shock. Eleven patients with Gram-positive infections including Streptococcus pneumoniae (n=7) and Staphylococcus aureus (n=4) and ten patients with Gram-negative infections including Klebsiella pneumoniae (n=4), Pseudomonas aeruginosa (n=3), Stenophomonas maltophilia (n=2), and Cupriavidus pauculus (n=1) were found. Antibiotic susceptibility testing revealed S. pneumoniae susceptible to penicillin, one case of S. aureus resistant to methicillin, three cases of K. pneumoniae resistant to third generation cephalosporin, and P. aeruginosa susceptible to ceftazidime. Mortality was 6 of 21 cases in which three pseudomonas cases were the initiation of inappropriate antibiotics. Conclusions: Gram-negative infections could cause bacteremia in pediatric patients with pneumonia and K. pneumoniae resistant to third generation cephalosporin and pseudomonas infection should be considered.

IntroductionSkeletal muscle has residential muscle stem cells, satellite cells (SC), which are indispensable for skeletal muscle regeneration and homeostasis. Previous studies highlighted the importance of SC microenvironment, or the niche, in controlling SC functions and fates. Among the SC niches, the neuronal inputs from motor neuron (MN) and neuromuscular junction (NMJ) are one of the indispensable factors for functional muscle regeneration after injury. Conversely, a recent study showed that depletion of SC could cause degeneration of NMJ, indicating that SCs also directly contribute to NMJ regeneration (Liu, 2017). However, the underlying mechanisms of SC‐MN communication during muscle regeneration are not well understood. Here, we hypothesized that the SC‐MN interaction enhances regeneration of damaged muscle by increasing myogenesis, protein synthesis, and mitochondrial function.Materials and MethodsGeorgia Tech Animal Care and Use Committee had reviewed and approved all animal procedures. Denervation (DEN) was performed on the sciatic nerves of young (3~5 months) and old (20~ months) C57BL/6, as well as mdx (a mouse model of DMD) by pinching them. The contralateral side was served as the control. SCs were isolated by FACS from DEN and the control muscles to compare RNA and protein expressions, proliferation, myogenesis, and cellular bioenergetics. Moreover, the in vivo regeneration and SC transplant capacities were tested.Results and DiscussionThe Wallerian degeneration of MN was evident up to post‐DEN day 7 (Figure 1a). Although the DEN muscle weight was decreased, the number of SC increased by 1.5‐fold at post‐DEN day 7 in young muscle (Figure 1b). In addition to the number of the SCs, we observed significant alteration of gene expression patterns after DEN. Specifically, genes associated with SC markers, cell cycle, and myogenesis were up‐regulated in young DEN muscle. In parallel, we observed significantly enhanced myogenesis of the SCs, which was also confirmed in vitro by proliferation, differentiation, and myogenic colony formation. As the myogenesis is energy demanding process, it was accompanied with the increased mitochondrial bioenergetics. Furthermore, the synergistic effect of the SC‐MN interaction was supported by increased in vivo regeneration after muscle/nerve injury and engraftment of transplanted SC. Interestingly, SC‐MN synergy observed in young was abolished in aged or mdx which have fragmented NMJs. It is suggesting that the lack of the regenerative capacity in these muscles is partially due to the abnormality in SC‐MN interaction (Figure 1b and c).ConclusionAll in all, our data show that the SC‐MN interaction synergistically enhances functional regeneration after skeletal muscle injury and depletion of this crosstalk may cause the lack of regeneration as shown in aged and mdx muscle.Support or Funding InformationS&R Foundation Ryuji Ueno Award (YCJ), NIH R21AR072287 (YCJ)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Model-based estimation often uses impact factors and historical data to predict the effort of new projects. Estimation accuracy of this approach is highly dependent on how well impact factors are selected. This paper comparatively assesses six methods for prune parameters of effort estimation models, including Stepwise regression, Lasso, constrained regression, GRASP, Tabu search, and PCA. Four data sets were used for evaluation, showing that estimation accuracy varies among the methods but no method consistently outperforms the rest. Stepwise regression prunes estimation model parameters the most while it does not sacrifice much estimation performance. Our study provides further evidence to support the use of Stepwise regression for selecting factors in effort estimation.

Peripheral artery disease (PAD) is a significant medical condition caused by blockages in the arteries of the leg. Some PAD patients progress to critical limb ischemia (CLI) and major amputation. While recent regenerative medicine approaches on collateral vessel formation have made some progress, the myopathy and dysregulation of the skeletal muscle in CLI have not been thoroughly investigated. PURPOSE: To determine the regenerative mechanism of the muscle stem cell (MuSC) and its niche components in response to ischemic insults, we assessed interactions between MuSC, vascular- and neural-network, and myofibers at different times points. METHODS: The femoral artery ligation mouse model of PAD on different reporter mice were used in the study. Immunofluorescence, single fiber staining, and biochemistry blotting from harvested hindlimb muscles were used for data analysis. One-way ANOVA with Tukey’s post hoc test and a paired two-tailed t-test were performed to determine differences following CLI injury. RESULTS: Skeletal muscle regeneration persisted up to 56 days while the number of eMHC+ fibers (p<0.01) was highest 14 days following CLI surgery compared to the contralateral sham control. In addition, muscle regeneration was accompanied by significant alterations in the motor unit, as demarcated by the presence of denervated synapses, regeneration of the neuromuscular junction (NMJ), and increased number of subsynaptic nuclei (p<0.05). Furthermore, the size of the myonuclear domain was decreased at 7 and 14 days (p<0.01), corresponding to greater RNA content (p<0.001) and MuSC frequency (p<0.05) while the mitochondrial domain was increased 28 days (p<0.01) following CLI injury. CONCLUSION: Overall, these data indicate that as a regenerative response to critical limb ischemia, the neurovascular network of myofibers are remodeled and newly regenerated myofibers exhibit MuSC-derived myonuclear expansion to allow enhanced transcriptional support and an increase in mitochondrial content for a bioenergetic need of the energy-demanding tissue regeneration. Supported by NIH R21AR072287 (YCJ) and Regenerative Engineering and Medicine research grant.

Critical limb ischemia, the most severe form of peripheral artery disease, leads to extensive damage and alterations to skeletal muscle homeostasis. Although recent research has investigated the tissue-specific responses to ischemia, the role of the muscle stem cell in the regeneration of its niche components within skeletal muscle has been limited. To elucidate the regenerative mechanism of the muscle stem cell in response to ischemic insults, we explored cellular interactions between the vasculature, neural network, and muscle fiber within the muscle stem cell niche. Using a surgical murine hindlimb ischemia model, we first discovered a significant increase in subsynaptic nuclei and remodeling of the neuromuscular junction following ischemia-induced denervation. In addition, ischemic injury causes significant alterations to the myofiber through a muscle stem cell-mediated accumulation of total myonuclei and a concomitant decrease in myonuclear domain size, possibly to enhance the transcriptional and translation output and restore muscle mass. Results also revealed an accumulation of total mitochondrial content per myonucleus in ischemic myofibers to compensate for impaired mitochondrial function and high turnover rate. Taken together, the findings from this study suggest that the muscle stem cell plays a role in motor neuron reinnervation, myonuclear accretion, and mitochondrial biogenesis for skeletal muscle regeneration following ischemic injury.

Loss-of-function mutations of the tassel-less1 (tls1) gene in maize, which is the co-ortholog of the Arabidopsis boron (B) importer NIP5;1, leads to the loss of reproductive structures (tassels and ears). The tls1 phenotypes can be rescued by B supplementation in the field and in the greenhouse. As the rescue with B supplementation is variable in the field, we investigated additional abiotic factors, potentially causing this variation in controlled greenhouse conditions. We found that the B-dependent rescue of the tls1 mutant tassel phenotype was enhanced when plants were grown with a mix of high pressure sodium (HPS) and metal halide (MH) lamps. Normal and tls1 plants had a significant increase in transpiration and increased B content in the leaves in the greenhouse with the addition of MH lamps. Our findings imply that B transport to the shoot is enhanced through increased transpiration, which suggests that the xylem transpiration stream provides a significant supply of B in maize.

Superoxide (Or) and other reactive oxygen species (ROS) are generated in response to numerous biotic and abiotic stresses. Different ROS have been reported to elicit different transcriptional responses in plants, and so ROS-responsive marker genes and promoter::reporter gene fusions have been proposed as indirect means of detecting ROS and discriminating among different species. However, further information about the specificity of transcriptional responses to O-2(-) is needed in order to assess potential markers for this critical stress-responsive signaling molecule. Using qRT-PCR, the expression of 12 genes previously reported to be upregulated by O-2(-) was measured in Arabidopsis thaliana plants exposed to elicitors of common stress-responsive ROS: methyl viologen (an inducer of O-2(-)), rose bengal (an inducer of singlet oxygen, (1)Delta O-2), and exogenous hydrogen peroxide (H2O2). Surprisingly, Zinc-Finger Protein 12 (AtZAT12), which had previously been used as a reporter for H2O2, responded more strongly to O-2(-) than to H2O2; moreover, the expression of an AtZAT12 promoter-reporter fusion (AtZAT12::Luc) was enhanced by diethyldithiocarbamate, which inhibits dismutation of O-2(-) to H2O2. These results suggest that AtZAT12 is transcriptionally upregulated in response to of, and that AtZAT12::Luc may be a useful biosensor for detecting O-2(-) generation in vivo. In addition, transcripts encoding uncoupling proteins (AtUCPs) showed selectivity for O-2(-) in Arabidopsis, and an AtUCP homolog upregulated by methyl viologen was also identified in maize (Zea mays L), indicating that there are O-2(-)- responsive members of this family in monocots. These results expand our limited knowledge of ROS-responsive gene expression in monocots, as well as O-2(-)-selective responses in dicots. (C) 2017 The Authors. Published by Elsevier Masson SAS.

After the successful cloning of the first gene for a polyglutamine disease in 1991, the expanded polyglutamine tract in the nine polyglutamine disease proteins became an obvious therapeutic target. Early hypotheses were that misfolded, precipitated protein could be a universal pathogenic mechanism. However, new data are accumulating on Huntington’s disease and other polyglutamine diseases that appear to contradict the toxic aggregate hypothesis. Recent data suggest that the toxic species of protein in these diseases may be soluble mutant conformers, and that the protein context of expanded polyglutamine is critical to understanding disease specificity. Here we discuss recent publications that define other important therapeutic targets for polyglutamine‐mediated neurodegeneration related to the context of the expanded polyglutamine tract in the disease protein.