Coordinated Activation of ARF1 GTPases by ARF-GEF GNOM Dimers Is Essential for Vesicle Trafficking in Arabidopsis.

Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

Cytokinin-induced promotion of root meristem size in the fern Azolla supports a shoot-like origin of euphyllophyte roots.

The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises the questions of how auxin and cytokinin govern fern root system architecture and whether this can tell us something about the origin of that root. Using Azolla filiculoides, we characterized the influence of IAA and zeatin on adventitious fern root meristems and vasculature by Nomarski microscopy. Simultaneously, RNAseq analyses, yielding 36,091 contigs, were used to uncover how the phytohormones affect root tip gene expression. We show that auxin restricts Azolla root meristem development, while cytokinin promotes it; it is the opposite effect of what is observed in Arabidopsis. Global gene expression profiling uncovered 145 genes significantly regulated by cytokinin or auxin, including cell wall modulators, cell division regulators and lateral root formation coordinators. Our data illuminate both evolution and development of fern roots. Promotion of meristem size through cytokinin supports the idea that root meristems of euphyllophytes evolved from shoot meristems. The foundation of these roots was laid in a postembryonically branching shoot system.

Nitric oxide prevents axonal degeneration by inducing HIF-1-dependent expression of erythropoietin.

Nitric oxide (NO) is a signaling molecule that can trigger adaptive (physiological) or maladaptive (pathological) responses to stress stimuli in a context-dependent manner. We have previously reported that NO may signal axonal injury to neighboring glial cells. In this study, we show that mice deficient in neuronal nitric oxide synthase (nNOS-/-) are more vulnerable than WT mice to toxin-induced peripheral neuropathy. The administration of NO donors to primary dorsal root ganglion cultures prevents axonal degeneration induced by acrylamide in a dose-dependent manner. We demonstrate that NO-induced axonal protection is dependent on hypoxia-inducible factor (HIF)-1-mediated transcription of erythropoietin (EPO) within glial (Schwann) cells present in the cultures. Transduction of Schwann cells with adenovirus AdCA5 encoding a constitutively active form of HIF-1α results in amelioration of acrylamide-induced axonal degeneration in an EPO-dependent manner. Mice that are partially deficient in HIF-1α (HIF-1α+/-) are also more susceptible than WT littermates to toxic neuropathy. Our results indicate that NO→HIF-1→EPO signaling represents an adaptive mechanism that protects against axonal degeneration.

Time to traumatic intracranial hematoma evacuation: contemporary standard and room for improvement.

PURPOSE: Traumatic intracranial hematoma (TICH) is a neurosurgical emergency with high mortality and morbidity. The time to operative decompression is a modifiable but inconsistently reported risk factor for TICH patients? OUTCOMES: We aimed to provide contemporary time to evacuation data and long-term trends in timing of TICH evacuation in a trauma system. METHODS: A 13-year retrospective cohort study ending in 2021 at a trauma system with one level-1 trauma center included all patients undergoing urgent craniotomy or craniectomy for evacuation of TICH. Demographics, injury severity and key timeframes of care were collected. Subgroups analyzed were polytrauma versus isolated head injury, direct admissions versus transfers and those who survived versus those who died. Linear regression of times from injury to operating room was performed. RESULTS: Seventy-eight TICH patients (Age: 35 (22-56); 58 (74%) males; ISS: 25(25-41); AIS head: 5 (4-5); mortality: 21 (27%) patients) were identified. Initial GCS was 8 (3.25-14) which decreased to 3 (3-7) by arrival in the trauma center. There were 46 (59%) patients intubated prior to arrival. Median time from injury to operation was 4.88 (3.63-6.80) hours. Linear regression of injury to OR showed increasing times to operative intervention for direct admissions to the trauma center over the study period (p=0.04). There was no associated change in mortality or Glasgow outcome score over the same time. CONCLUSION: This contemporary data shows timing from injury to evacuation is approaching 5 hours. Over the 13-year study period the time to operative intervention significantly increased for direct admissions. This study will guide our institutions response to TICH presentations in the future. Other trauma systems should critically appraise their results with the same reporting standard.

Effect of a Garlic and Citrus Extract Supplement on the Lactation Performance and Carbon Footprint of Dairy Cows under Grazing Conditions in Chile.

Two trials were conducted to evaluate the effect of a garlic and citrus extract supplement (GCE) on the milk production performance and carbon footprint of grazing dairy cows in a Chilean commercial farm. A total of 36 early- to mid-lactation and 54 late-lactation Irish Holstein-Friesian cows were used in Trial 1 and Trial 2, respectively. In both trials, the cows were reared under grazing conditions and offered a supplementary concentrate without or with GCE (33 g/cow/d) for 12 weeks. The concentrate was fed in the afternoon when the cows visited the milking parlour. Consequently, the results of milk production performance in these trials were used to determine the effect of feeding with GCE on the carbon footprint (CFP) of milk using a life cycle assessment (LCA) model. In Trial 1 and Trial 2, feeding with GCE increased estimated dry matter intake (DMI, kg/d) by 8.15% (18.4 vs. 19.9) and 15.3% (15.0 vs. 17.3), energy-corrected milk (ECM, kg/d) by 11.4% (24.5 vs. 27.3) and 33.5% (15.5 vs. 20.7), and feed efficiency (ECM/DMI) by 3.03% (1.32 vs. 1.36) and 17.8% (1.01 vs. 1.19), respectively. The LCA revealed that feeding with GCE reduced the emission intensity of milk by 8.39% (1.55 vs. 1.42 kg CO2-eq/kg ECM). Overall, these results indicate that feeding with GCE improved the production performance and CFP of grazing cows under the conditions of the current trials.

The Australian Trauma Registry (ATR): a leading clinical quality registry.

Operating since 2012 under the auspices of the Australian Trauma Quality Improvement Program (AusTQIP), the Australian Trauma Registry (ATR) has established itself as a leading clinical quality registry (CQR). Initially developed as a national program for improved safety and quality trauma care across Australian trauma centers, it has since expanded to include New Zealand, becoming one of the few bi-national trauma registries. The registry has recorded close to 100,000 episodes of care for severely injured patients since its inception, with 10.7% growth in annual inclusions. The ATR, administered by the National Trauma Research Institute (NTRI), monitors the continuum of trauma care from pre-hospital settings, to discharge from definitive care. Collection and analysis of data about severely injured trauma patients, their injuries, management and outcomes, aims to inform future improvements to health service provision and reduce preventable morbidity and mortality.

The epidemiology of overtransfusion of red cells in trauma resuscitation patients in the context of a mature massive transfusion protocol.

PURPOSE: Packed red blood cell (PRBC) transfusion remains an integral part of trauma resuscitation and an independent predictor of unfavourable outcomes. It is often administered urgently based on clinical judgement. These facts put trauma patients at high risk of potentially dangerous overtransfusion. We hypothesised that trauma patients are frequently overtransfused and overtransfusion is associated with worse outcomes. METHODS: Trauma patients who received PRBCs within 24 h of admission were identified from the trauma registry during the period January 1 2011-December 31 2018. Overtransfusion was defined as haemoglobin concentration of greater than or equal to 110 g/L at 24 h post ED arrival (± 12 h). Demographics, injury severity, injury pattern, shock severity, blood gas values and outcomes were compared between overtransfused and non-overtransfused patients. RESULTS: From the 211 patients (mean age 45 years, 71% male, ISS 27, mortality 12%) who met inclusion criteria 27% (56/211) were overtransfused. Patients with a higher pre-hospital systolic blood pressure (112 vs 99 mmHg p < 0.01) and a higher initial haemoglobin concentration (132 vs 124 p = 0.02) were more likely to be overtransfused. Overtransfused patients received smaller volumes of packed red blood cells (5 vs 7 units p = 0.049), fresh frozen plasma (4 vs 6 units p < 0.01) and cryoprecipitate (6 vs 9 units p = 0.01) than non-overtransfused patients. CONCLUSION: More than a quarter of patients in our cohort were potentially given more blood products than required without obvious clinical consequences. There were no clinically relevant associations with overtransfusion.

Is sphincter preservation reasonable in all patients with rectal cancer?

PURPOSE: Modern sphincter-preserving surgery for ultralow rectal carcinoma has a comparable oncological radicality to abdomino-perineal extirpation (APE). The aim of this study was to assess the long-term morbidity of ultralow anterior resection (ULAR) and its impact on quality of life (QoL) METHODS: The medical records of 142 consecutive patients who underwent surgery for ultralow rectal carcinoma from January 1991 to December 2004 were reviewed retrospectively. The rate of rehospitalisation and rate of non-reversed temporary stomas ("failure" stoma) were analysed. Generic and cancer-specific quality of life questionnaires were used to assess quality of life. RESULTS: There were a total of 82 ULAR and 60 APE. After ULAR, 25 (30.5%) of the patients were readmitted, stenosis and anastomotic leakage being the main reasons. After APE, only 2 (3.3%) of the patients were readmitted (P < 0.001). The rate of patients with a permanent stoma after sphincter-saving surgery was 22.0%. The failure rate was higher for older patients (P = 0.005) and for coloanal pull-through anastomosis (P = 0.001). The exploratory analysis revealed a negative impact of a "failure" stoma on QoL. CONCLUSION: Severe long-term morbidity and high failure rate of stoma reversal have a significantly worse impact on QoL after ULAR; therefore, APE is a valid alternative to ULAR, especially in elder patients with planned coloanal pull-through anastomosis.

Spatially distinct and functionally independent mechanisms of axonal degeneration in a model of HIV-associated sensory neuropathy.

Sensory polyneuropathies are the most frequent neurological complication of human immunodeficiency virus (HIV) infection. Distal symmetric polyneuropathy (DSP), associated with HIV infection, is characterized by length-dependent axonal degeneration of sensory fibres. In rodent dorsal root ganglia (DRG) cultures, HIV viral envelope protein gp120 results in neurotoxicity and axonal degeneration. Since it is unknown whether the axonal degeneration is a consequence of neuronal death or whether it is due to a direct toxic effect on axons, we investigated gp120-induced axonal toxicity using compartmentalized cultures of sensory neurons. Our results show that gp120 causes neuronal apoptosis and axonal degeneration through two, independent and spatially separated mechanisms of action: (i) an indirect insult to cell bodies, requiring the presence of Schwann cells, results in neuronal apoptotic death and subsequent axonal degeneration; (ii) a direct, local toxicity exerted on axons through activation of mitochondrial caspase pathway that is independent of cell body. This local axonal toxicity is mediated through binding of gp120 to axonal chemokine receptors and can be prevented by chemokine receptor blockers. In conclusion, we propose a novel pathway of axonal degeneration mediated by gp120 that is dependent on local activation of caspases in the axon. This observation suggests that axonal protection is a relevant therapeutic target for HIV-associated sensory neuropathy. Furthermore, chemokine receptor inhibitors, which are currently being developed as HIV entry inhibitor drugs, may also have a therapeutic role in HIV-associated peripheral neuropathies by preventing axonal degeneration.

Differences between hospitals in cesarean rates for term primigravidas with cephalic presentation.

OBJECTIVE: The purpose of this study was to quantify patient populations and practice patterns at perinatal centers with the highest and lowest cesarean delivery rates. METHODS: The 2 perinatal centers in our state with the lowest (Hospital A-16.6%) and highest (Hospital B-20.3%) overall cesarean rates for Robson group 1 (term primigravidas, vertex, spontaneous labor) and group 2 (term primigravidas, vertex, induced labor) were identified. A total of 174 medical records at Hospital A and 150 records at Hospital B were reviewed. Statistical analysis was performed using independent-sample t tests, chi(2), and multiple logistic regression. RESULTS: Indications for cesarean delivery were not different between the 2 groups, with the majority being for failure to progress in labor and nonreassuring fetal status. There were no differences between groups in rates of postpartum hemorrhage, chorioamnionitis, or endometritis. There were no differences in neonatal outcomes. Although women delivering in hospital A were not more likely to receive oxytocin augmentation (P = .291), their mean maximal oxytocin dosage was higher (14.5 units compared with 11.6 units, P < .001), and they were more likely to receive both fetal scalp electrodes (60.9% compared with 37.3%, P < .001) and intrauterine pressure catheters (63.8% compared with 26.0%, P < .001). CONCLUSION: Because safe reduction in cesarean delivery rates for primigravidas will proportionately reduce the number of repeat cesarean delivery required, benchmarking practices as described in this study can be considered in obstetric practices interested in long-term reductions of their cesarean delivery rates. LEVEL OF EVIDENCE: III.

The Tec family of tyrosine kinases in T cells, amplifiers of T cell receptor signals.

ITK and Rlk/Txk are the predominant Tec family of tyrosine kinases expressed in T cells, and are involved in T cell antigen receptor mediated activation of T cells. These kinases require prior activation of Lck, Zap-70 and PI3-kinase for efficient activation. They share major substrates with both Lck and Zap-70, however the pathways they regulate are unclear. Recent evidence suggests that these kinases may not activate unique pathways, but instead serve as amplifiers for the upstream kinases Lck and Zap-70. This review will discuss the evidence for this view.

Differential sensitivity to Itk kinase signals for T helper 2 cytokine production and chemokine-mediated migration.

Allergic asthma is dependent on chemokine-mediated Th2 cell migration and Th2 cytokine secretion into the lungs. The inducible T cell tyrosine kinase Itk regulates the production of Th2 cytokines as well as migration in response to chemokine gradients. Mice lacking Itk are resistant to developing allergic asthma. However, the role of kinase activity of Itk in the development of this disease is unclear. In addition, whether distinct Itk-derived signals lead to T cell migration and secretion of Th2 cytokines is also unknown. Using transgenic mice specifically lacking Itk kinase activity, we show that active kinase signaling is required for control of Th2 responses and development of allergic asthma. Moreover, dominant suppression of kinase Itk activity led to normal Th2 responses, but significantly reduced chemokine-mediated migration, resulting in prevention of allergic asthma. These observations indicate that signals required for Th2 responses and migration are differentially sensitive to Itk activity. Manipulation of Itk's activity can thus provide a new strategy to treat allergic asthma by differentially affecting migration of T cells into the lungs, leaving Th2 responses intact.

Regulation of CXC chemokine receptor 4-mediated migration by the Tec family tyrosine kinase ITK.

Chemokines are critical in controlling lymphocyte traffic and migration. The CXC chemokine CXCL12/SDF-1alpha interacts with its receptor CXCR4 to induce the migration of a number of different cell types. Although an understanding of the physiological functions of this chemokine is emerging, the mechanism by which it regulates T cell migration is still unclear. We show here that the Tec family kinase ITK is activated rapidly following CXCL12/SDF-1alpha stimulation, and this requires Src and phosphatidylinositol 3-kinase activities. ITK regulates the ability of CXCL12/SDF-1alpha to induce T cell migration as overexpression of wild-type ITK-enhanced migration, and T cells lacking ITK exhibit reduced migration as well as adhesion in response to CXCL12/SDF-1alpha. Further analysis suggests that ITK may regulate CXCR4-mediated migration and adhesion by altering the actin cytoskeleton, as ITK null T cells were significantly defective in CXCL12/SDF-1a-mediated actin polymerization. Our data suggest that ITK may regulate the ability of CXCR4 to induce T cell migration.

A novel endogenous erythropoietin mediated pathway prevents axonal degeneration.

Clinically relevant peripheral neuropathies (such as diabetic and human immunodeficiency virus sensory neuropathies) are characterized by distal axonal degeneration, rather than neuronal death. Here, we describe a novel, endogenous pathway that prevents axonal degeneration. We show that in response to axonal injury, periaxonal Schwann cells release erythropoietin (EPO), which via EPO receptor binding on neurons, prevents axonal degeneration. We demonstrate that the relevant axonal injury signal that stimulates EPO production from surrounding glial cells is nitric oxide. In addition, we show that this endogenous pathway can be therapeutically exploited by administering exogenous EPO. In an animal model of distal axonopathy, systemic EPO administration prevents axonal degeneration, and this is associated with a reduction in limb weakness and neuropathic pain behavior. Our in vivo and in vitro data suggest that EPO prevents axonal degeneration and therefore may be therapeutically useful in a wide variety of human neurological diseases characterized by axonopathy.