Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis.

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (MlklFLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8-/-MlklFLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8+/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8-/-MlklFLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

Topical tranexamic acid (TXA) is non-inferior to intravenous TXA in adult spine surgery: a meta-analysis.

Tranexamic acid (TXA) has long been utilized in spine surgery and can be administered through intravenous (IV) and topical routes. Although, topical and IV administration of TXA are both effective in decreasing blood loss during spine surgery, complications like deep vein thrombosis (DVT) and pulmonary embolism have been reported with the use of intravenous TXA (ivTXA). These potential complications may be mitigated through the use of topical TXA (tTXA). To assess optimal dosing protocols and efficacy of topical TXA in spine surgery, Embase, Ovid-MEDLINE, Scopus, Cochrane, and clinicaltrials.gov were queried for original research on the use of tTXA in adult patients undergoing spine surgery. Data parameters analyzed included blood loss, transfusion rate, thromboembolic, and other complications. Data was synthesized and confidence evaluated according to the Grades of Recommendation, Assessment, Development, and Evaluation approach. Nineteen studies were included in the final analysis with 2197 patients. Of the 18 published studies, 9 (50%) displayed high levels of evidence. Topical TXA showed a trend towards a lower risk of transfusion and complications. Protocols that used 1g tTXA showed a significantly reduced risk for transfusion when compared to controls (risk ratio -1.05, 95% CI (-1.62, -0.48); P = 0.94, I2 = 0%). Complications associated with tTXA included DVTs and wound infections. Topical TXA was non-inferior to intravenous TXA with similar efficacy and complication profiles for bleeding control in spine surgery; however, more studies are needed to discern benefits and risks.

Diagnostic utility of different types of somatosensory evoked potential changes in pediatric idiopathic scoliosis correction surgery.

PURPOSE: To evaluate the diagnostic accuracy of intraoperative somatosensory evoked potential (SSEP) monitoring and types of SSEP changes in predicting the risk of postoperative neurological outcomes during correction surgery for idiopathic scoliosis (IS) in the pediatric age group (≤ 21 years). METHODS: Database review was performed to identify literature on pediatric patients with IS who underwent correction with intraoperative neuromonitoring. The sensitivity, specificity, and diagnostic odds ratio (DOR) of transient and persistent SSEP changes and complete SSEP loss in predicting postoperative neurological deficits were calculated. RESULTS: Final analysis included 3778 patients. SSEP changes had a sensitivity of 72.9%, specificity of 96.8%, and DOR of 102.3, while SSEP loss had a sensitivity of 41.8%, specificity of 99.3%, and DOR of 133.2 for predicting new neurologic deficits. Transient and persistent SSEP changes had specificities of 96.8% and 99.1%, and DORs of 16.6 and 59, respectively. CONCLUSION: Intraoperative SSEP monitoring can predict perioperative neurological injury and improve surgical outcomes in pediatric scoliosis fusion surgery. LEVEL OF EVIDENCE: Level 2. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Neck Symptoms and Associated Clinical Outcomes in Patients Following Concussion.

OBJECTIVE: To examine the frequency and association of neck pain symptoms in patients with a concussion. STUDY SETTING AND PARTICIPANTS: Three-hundred and thirty-one consecutively enrolled patients aged 9 to 68 years with a diagnosed concussion 1 to 384 days post-injury were enrolled at a concussion clinic from a single integrated healthcare system in Western Pennsylvania between 2019 and 2021. DESIGN: Retrospective cohort analysis of prospectively collected concussion screening tool intake survey responses and clinical outcomes data. The primary outcome was self-reported neck pain or difficulty with neck movement on the Concussion Clinical Profiles Screening (CP Screen) tool, recovery time, and incidence of treatment referral. Immediate Post-concussion Assessment and Cognitive Testing (ImPACT) composite scores, Vestibular/Ocular Motor Screening (VOMS) item scores, type and severity of neck symptoms, mechanism of injury, time from injury to clinic presentation, medical history, and concussion symptom profile were secondary outcomes. RESULTS: Of the 306 consecutively enrolled eligible patients in the registry, 145 (47%) reported neck pain, 68 (22.2%) reported difficulty moving their neck, and 146 (47.7%) reported either symptom. A total of 47 (15.4%) participants reported more severe neck symptoms, and this group took longer to recover (40 ± 27 days) than those not reporting neck symptoms (30 ± 28 days; U = 8316, P < .001). Stepwise logistic regression predicting more severe neck symptoms was significant (Nagelkerke R2 = 0.174, χ 2 = 9.315, P = .316) with older age ( P = .019) and mechanism of injury including motor vehicle collisions (MVCs) ( P = .047) and falls ( P = .044) as risk factors. MVCs and falls were associated with over 4 times and 2 times greater risk, respectively, for reporting more severe neck symptoms. CONCLUSION: Neck pain and stiffness symptoms are common in patients with a concussion following high-energy mechanisms of injury including MVCs or falls from height. These symptoms are associated with prolonged recovery. Providers should evaluate neck symptoms and consider targeted treatment strategies to limit their effects in patients with a concussion.

Utility of transcranial motor-evoked potential changes in predicting postoperative deficit in lumbar decompression and fusion surgery: a systematic review and meta-analysis.

PURPOSE: The primary aim of this study was to evaluate whether TcMEP alarms can predict the occurrence of postoperative neurological deficit in patients undergoing lumbar spine surgery. The secondary aim was to determine whether the various types of TcMEP alarms including transient and persistent changes portend varying degrees of injury risk. METHODS: This was a systematic review and meta-analysis of the literature from PubMed, Web of Science, and Embase regarding outcomes of transcranial motor-evoked potential (TcMEP) monitoring during lumbar decompression and fusion surgery. The sensitivity, specificity, and diagnostic odds ratio (DOR) of TcMEP alarms for predicting postoperative deficit were calculated and presented with forest plots and a summary receiver operating characteristic curve. RESULTS: Eight studies were included, consisting of 4923 patients. The incidence of postoperative neurological deficit was 0.73% (36/4923). The incidence of deficits in patients with significant TcMEP changes was 11.79% (27/229), while the incidence in those without changes was 0.19% (9/4694). All TcMEP alarms had a pooled sensitivity and specificity of 63 and 95% with a DOR of 34.92 (95% CI 7.95-153.42). Transient and persistent changes had sensitivities of 29% and 47%, specificities of 96% and 98%, and DORs of 8.04 and 66.06, respectively. CONCLUSION: TcMEP monitoring has high specificity but low sensitivity for predicting postoperative neurological deficit in lumbar decompression and fusion surgery. Patients who awoke with new postoperative deficits were 35 times more likely to have experienced TcMEP changes intraoperatively, with persistent changes indicating higher risk of deficit than transient changes. LEVEL OF EVIDENCE II: Diagnostic Systematic Review.

Multilevel tandem spondylolisthesis associated with a reduced "safe zone" for a transpsoas lateral lumbar interbody fusion at L4-5.

OBJECTIVE: The aim of this study was to investigate the effect of degenerative spondylolisthesis (DS) on psoas anatomy and the L4-5 safe zone during lateral lumbar interbody fusion (LLIF). METHODS: In this retrospective, single-institution analysis, patients managed for low-back pain between 2016 and 2021 were identified. Inclusion criteria were adequate lumbar MR images and radiographs. Exclusion criteria were spine trauma, infection, metastases, transitional anatomy, or prior surgery. There were three age and sex propensity-matched cohorts: 1) controls without DS; 2) patients with single-level DS (SLDS); and 3) patients with multilevel, tandem DS (TDS). Axial T2-weighted MRI was used to measure the apical (ventral) and central positions of the psoas relative to the posterior tangent line at the L4-5 disc. Lumbar lordosis (LL), pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), and PI-LL mismatch were measured on lumbar radiographs. The primary outcomes were apical and central psoas positions at L4-5, which were calculated using stepwise multivariate linear regression including demographics, spinopelvic parameters, and degree of DS. Secondary outcomes were associations between single- and multilevel DS and spinopelvic parameters, which were calculated using one-way ANOVA with Bonferroni correction for between-group comparisons. RESULTS: A total of 230 patients (92 without DS, 92 with SLDS, and 46 with TDS) were included. The mean age was 68.0 ± 8.9 years, and 185 patients (80.4%) were female. The mean BMI was 31.0 ± 7.1, and the mean age-adjusted Charlson Comorbidity Index (aCCI) was 4.2 ± 1.8. Age, BMI, sex, and aCCI were similar between the groups. Each increased grade of DS (no DS to SLDS to TDS) was associated with significantly increased PI (p < 0.05 for all relationships). PT, PI-LL mismatch, center psoas, and apical position were all significantly greater in the TDS group than in the no-DS and SLDS groups (p < 0.05). DS severity was independently associated with 2.4-mm (95% CI 1.1-3.8 mm) center and 2.6-mm (95% CI 1.2-3.9 mm) apical psoas anterior displacement per increased grade (increasing from no DS to SLDS to TDS). CONCLUSIONS: TDS represents more severe sagittal malalignment (PI-LL mismatch), pelvic compensation (PT), and changes in the psoas major muscle compared with no DS, and SLDS and is a risk factor for lumbar plexus injury during L4-5 LLIF due to a smaller safe zone.

Examining the safety profile of a standard dose tranexamic acid regimen in spine surgery.

OBJECTIVE: Perioperative blood loss during spinal surgery is associated with complications and in-hospital mortality. Weight-based administration of tranexamic acid (TXA) has the potential to reduce blood loss and related complications in spinal surgery; however, evidence for standardized dosing is lacking. The purpose of this study was to evaluate the impact of a standardized preoperative 2 g bolus TXA dosing regimen on perioperative transfusion, blood loss, thromboembolic events, and postoperative outcomes in spine surgery patients. METHODS: An institutional review board approved this retrospective review of prospectively enrolled adult spine patients (> 18 years of age). Patients were included who underwent elective and emergency spine surgery between September 2018 and July 2021. Patients who received a standardized 2 g dose of TXA were compared to patients who did not receive TXA. The primary outcome measure was perioperative transfusion. Secondary outcomes included estimated blood loss and thromboembolic or other perioperative complications. Descriptive statistics were calculated, and continuous variables were analyzed with the two-tailed independent t-test, while categorical variables were analyzed with the Fisher's exact test or chi-square test. Stepwise multivariate regression analysis was performed to examine independent risk factors for perioperative outcomes. RESULTS: TXA was administered to 353 of 453 (78%) patients, and there were no demographic differences between groups. Although the TXA group had more operative levels and a longer operative time, the transfusion rate was not different between the TXA and no-TXA groups (7.4% vs 8%, p = 0.83). Stepwise multivariate regression found that the number of operative levels was an independent predictor of perioperative transfusion and that both operative levels and operative time were correlated with estimated blood loss. TXA was not identified as an independent predictor of any postoperative complication. CONCLUSIONS: A standardized preoperative 2 g bolus TXA dosing regimen was associated with an excellent safety profile, and despite increased case complexity in terms of number of operative levels and operative time, patients treated with TXA did not require more blood transfusions than patients not treated with TXA.

How Do PROMIS Scores Correspond to Common Physical Abilities?

BACKGROUND: The Patient-Report Outcomes Measurement Information System (PROMIS) is increasingly used as a general-purpose tool for measuring orthopaedic surgery outcomes. This set of questionnaires is efficient, precise, and correlates well with specialty-specific measures, but impactful implementation of patient-specific data, especially at the point of care, remains a challenge. Although clinicians may have substantial experience with established patient-reported outcome measures in their fields, PROMIS is relatively new, and the real-life meaning of PROMIS numerical summary scores may be unknown to many orthopaedic surgeons. QUESTIONS/PURPOSES: We aimed to (1) identify a small subset of important items in the PROMIS Physical Function (PF) item bank that are answered by many patients with orthopaedic conditions and (2) graphically display characteristic responses to these items across the physical function spectrum in order to translate PROMIS numerical scores into physical ability levels using clinically relevant, familiar terms. METHODS: In a cross-sectional study, 97,852 PROMIS PF assessments completed by 37,517 patients with orthopaedic conditions presenting to a tertiary-care academic institution were pooled and descriptively analyzed. Between 2017 and 2020, we evaluated 75,354 patients for outpatient orthopaedic care. Of these, 67% (50,578) were eligible for inclusion because they completed a PROMIS version 2.0 physical function assessment; 17% (12,720) were excluded because they lacked information in the database on individual item responses, and another < 1% (341) were excluded because the assessment standard error was greater than 0.32, leaving 50% of the patients (37,517) for analysis. The PROMIS PF is scored on a 0-point to 100-point scale, with a population mean of 50 and SD of 10. Anchor-based minimum clinically important differences have been found to be 8 to 10 points in a foot and ankle population, 7 to 8 points in a spine population, and approximately 4 points in a hand surgery population. The most efficient and precise means of administering the PROMIS PF is as a computerized adaptive test (CAT), whereby an algorithm intelligently tailors each follow-up question based on responses to previous questions, requiring only a few targeted questions to generate an accurate result. In this study, the mean PROMIS PF score was 41 ± 9. The questions most frequently used by the PROMIS CAT software were identified (defined in this study as any question administered to > 0.1% of the cohort). To understand the ability levels of patients based on their individual scores, patients were grouped into score categories: < 18, 20 ± 2, 25 ± 2, 30 ± 2, 35 ± 2, 40 ± 2, 45 ± 2, 50 ± 2, 55 ± 2, 60 ± 2, and > 62. For each score category, the relative frequency of each possible response (ranging from "cannot do" to "without any difficulty") was determined for each question. The distribution of responses given by each score group for each question was graphically displayed to generate an intuitive map linking PROMIS scores to patient ability levels (with ability levels represented by how patients responded to the PROMIS items). RESULTS: Twenty-eight items from the 165-question item bank were used frequently (that is, administered to more than 0.1% of the cohort) by the PROMIS CAT software. The top four items constituted 63% of all items. These top four items asked about the patient's ability to perform 2 hours of physical labor, yard work, household chores, and walking more than 1 mile. Graphical displays of responses to the top 28 and top four items revealed how PROMIS scores correspond to patient ability levels. Patients with a score of 40 most frequently responded that they experienced "some difficulty" with physical labor, yard work, household chores, and walking more than 1 mile, compared with "little" or "no" difficulty for patients with a score of 50 and "cannot do" for patients with a score of 30. CONCLUSION: We provided a visual key linking PROMIS numerical scores to physical ability levels using clinically relevant, familiar terms. Future studies might investigate whether using similar graphical displays as a patient education tool enhances patient-provider communication and improves the patient experience. CLINICAL RELEVANCE: The visual explanation of PROMIS scores provided by this study may help new users of the PROMIS understand the instrument, feel empowered to incorporate it into their practices, and use it as a tool for counseling patients about their scores.

Quantifying variation in female internal genitalia: no evidence for plasticity in response to sexual conflict risk in a seed beetle.

Sexually antagonistic coevolution can drive the evolution of male traits that harm females, and female resistance to those traits. While males have been found to vary their harmfulness to females in response to social cues, plasticity in female resistance traits remains to be examined. Here, we ask whether female seed beetles Callosobruchus maculatus are capable of adjusting their resistance to male harm in response to the social environment. Among seed beetles, male genital spines harm females during copulation and females might resist male harm via thickening of the reproductive tract walls. We develop a novel micro computed tomography imaging technique to quantify female reproductive tract thickness in three-dimensional space, and compared the reproductive tracts of females from populations that had evolved under high and low levels of sexual conflict, and for females reared under a social environment that predicted either high or low levels of sexual conflict. We find little evidence to suggest that females can adjust the thickness of their reproductive tracts in response to the social environment. Neither did evolutionary history affect reproductive tract thickness. Nevertheless, our novel methodology was capable of quantifying fine-scale differences in the internal reproductive tracts of individual females, and will allow future investigations into the internal organs of insects and other animals.

Glucosylceramide production maintains colon integrity in response to Bacteroides fragilis toxin-induced colon epithelial cell signaling.

Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human health due to its ability to induce colitis and cause colon tumor formation in mice through the production of B. fragilis toxin (BFT). The formation of tumors is dependent on a pro-inflammatory signaling cascade, which begins with the disruption of epithelial barrier integrity through cleavage of E-cadherin. Here, we show that BFT increases levels of glucosylceramide, a vital intestinal sphingolipid, both in mice and in colon organoids (colonoids) generated from the distal colons of mice. When colonoids are treated with BFT in the presence of an inhibitor of glucosylceramide synthase (GCS), the enzyme responsible for generating glucosylceramide, colonoids become highly permeable, lose structural integrity, and eventually burst, releasing their contents into the extracellular matrix. By increasing glucosylceramide levels in colonoids via an inhibitor of glucocerebrosidase (GBA, the enzyme that degrades glucosylceramide), colonoid permeability was reduced, and bursting was significantly decreased. In the presence of BFT, pharmacological inhibition of GCS caused levels of tight junction protein 1 (TJP1) to decrease. However, when GBA was inhibited, TJP1 levels remained stable, suggesting that BFT-induced production of glucosylceramide helps to stabilize tight junctions. Taken together, our data demonstrate a glucosylceramide-dependent mechanism by which the colon epithelium responds to BFT.

ISSLS prize in basic science 2021: a novel inducible system to regulate transgene expression of TIMP1.

PURPOSE: Inflammatory and oxidative stress upregulates matrix metalloproteinase (MMP) activity, leading to intervertebral disc degeneration (IDD). Gene therapy using human tissue inhibitor of metalloproteinase 1 (hTIMP1) has effectively treated IDD in animal models. However, persistent unregulated transgene expression may have negative side effects. We developed a recombinant adeno-associated viral (AAV) gene vector, AAV-NFκB-hTIMP1, that only expresses the hTIMP1 transgene under conditions of stress. METHODS: Rabbit disc cells were transfected or transduced with AAV-CMV-hTIMP1, which constitutively expresses hTIMP1, or AAV-NFκB-hTIMP1. Disc cells were selectively treated with IL-1ß. NFκB activation was verified by nuclear translocation. hTIMP1 mRNA and protein expression were measured by RT-PCR and ELISA, respectively. MMP activity was measured by following cleavage of a fluorogenic substrate. RESULTS: IL-1ß stimulation activated NFκB demonstrating that IL-1ß was a surrogate for inflammatory stress. Stimulating AAV-NFκB-hTIMP1 cells with IL-1ß increased hTIMP1 expression compared to unstimulated cells. AAV-CMV-hTIMP1 cells demonstrated high levels of hTIMP1 expression regardless of IL-1ß stimulation. hTIMP1 expression was comparable between IL-1ß stimulated AAV-NFκB-hTIMP1 cells and AAV-CMV-hTIMP1 cells. MMP activity was decreased in AAV-NFκB-hTIMP1 cells compared to baseline levels or cells exposed to IL-1ß. CONCLUSION: AAV-NFκB-hTIMP1 is a novel inducible transgene delivery system. NFκB regulatory elements ensure that hTIMP1 expression occurs only with inflammation, which is central to IDD development. Unlike previous inducible systems, the AAV-NFκB-hTIMP1 construct is dependent on endogenous factors, which minimizes potential side effects caused by constitutive transgene overexpression. It also prevents the unnecessary production of transgene products in cells that do not require therapy.

Glucosylceramidase Maintains Influenza Virus Infection by Regulating Endocytosis.

Influenza virus is an RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Previous studies showed that influenza virus infection depends on cellular lipids, including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza virus infection following clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR-Cas9)-mediated knockout (KO) of its metabolizing enzyme glucosylceramidase (GBA). After confirming GBA knockout of HEK 293 and A549 cells by both Western blotting and lipid mass spectrometry, we observed diminished infection in both KO cell lines by a PR8 (H1N1) green fluorescent protein (GFP) reporter virus. We further showed that the reduction in infection correlated with impaired influenza virus trafficking to late endosomes and hence with fusion and entry. To examine whether GBA is required for other enveloped viruses, we compared the results seen with entry mediated by the glycoproteins of Ebola virus, influenza virus, vesicular stomatitis virus (VSV), and measles virus in GBA knockout cells. Entry inhibition was relatively robust for Ebola virus and influenza virus, modest for VSV, and mild for measles virus, suggesting a greater role for viruses that enter cells by fusing with late endosomes. As the virus studies suggested a general role for GBA along the endocytic pathway, we tested that hypothesis and found that trafficking of epidermal growth factor (EGF) to late endosomes and degradation of its receptor were impaired in GBA knockout cells. Collectively, our findings suggest that GBA is critically important for endocytic trafficking of viruses as well as of cellular cargos, including growth factor receptors. Modulation of glucosylceramide levels may therefore represent a novel accompaniment to strategies to antagonize "late-penetrating" viruses, including influenza virus.IMPORTANCE Influenza virus is the pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza virus enters host cells may lead to the development of more-efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza virus trafficking to late endosomes and for consequent fusion, entry, and infection. We also provide evidence that promotion of influenza virus entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence for optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos, such as the epidermal growth factor (EGF) receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as for pathogenic processes, including virus entry and Gaucher disease.

The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size.

The physiological constraints on bud burst in woody perennials, including vascular development and oxygenation, remain unresolved. Both light and tissue oxygen status have emerged as important cues for vascular development in other systems; however, grapevine buds have only a facultative light requirement, and data on the tissue oxygen status have been confounded by the spatial variability within the bud. Here, we analysed apoplastic development at early stages of grapevine bud burst and combined molecular modelling with histochemical techniques to determine the pore size of cell walls in grapevine buds. The data demonstrate that quiescent grapevine buds were impermeable to apoplastic dyes (acid fuchsin and eosin Y) until after bud burst was established. The molecular exclusion size was calculated to be 2.1 nm, which would exclude most macromolecules except simple sugars and phytohormones until after bud burst. We used micro-computed tomography to demonstrate that tissue oxygen partial pressure data correlated well with structural heterogeneity of the bud and differences in tissue density, confirming that the primary bud complex becomes rapidly and preferentially oxygenated during bud burst. Taken together, our results reveal that the apoplastic porosity is highly regulated during the early stages of bud burst, suggesting a role for vascular development in the initial, rapid oxygenation of the primary bud complex.

A new method for mapping spatial resolution in compound eyes suggests two visual streaks in fiddler crabs.

Visual systems play a vital role in guiding the behaviour of animals. Understanding the visual information animals are able to acquire is therefore key to understanding their visually mediated decision making. Compound eyes, the dominant eye type in arthropods, are inherently low-resolution structures. Their ability to resolve spatial detail depends on sampling resolution (interommatidial angle) and the quality of ommatidial optics. Current techniques for estimating interommatidial angles are difficult, and generally require in vivo measurements. Here, we present a new method for estimating interommatidial angles based on the detailed analysis of 3D micro-computed tomography images of fixed samples. Using custom-made MATLAB software, we determined the optical axes of individual ommatidia and projected these axes into the 3D space around the animal. The combined viewing directions of all ommatidia, estimated from geometrical optics, allowed us to estimate interommatidial angles and map the animal's sampling resolution across its entire visual field. The resulting topographic representations of visual acuity match very closely the previously published data obtained from both fiddler and grapsid crabs. However, the new method provides additional detail that was not previously detectable and reveals that fiddler crabs, rather than having a single horizontal visual streak as is common in flat-world inhabitants, probably have two parallel streaks located just above and below the visual horizon. A key advantage of our approach is that it can be used on appropriately preserved specimens, allowing the technique to be applied to animals such as deep-sea crustaceans that are inaccessible or unsuitable for in vivo approaches.

Clusters of iron-rich cells in the upper beak of pigeons are macrophages not magnetosensitive neurons.

Understanding the molecular and cellular mechanisms that mediate magnetosensation in vertebrates is a formidable scientific problem. One hypothesis is that magnetic information is transduced into neuronal impulses by using a magnetite-based magnetoreceptor. Previous studies claim to have identified a magnetic sense system in the pigeon, common to avian species, which consists of magnetite-containing trigeminal afferents located at six specific loci in the rostral subepidermis of the beak. These studies have been widely accepted in the field and heavily relied upon by both behavioural biologists and physicists. Here we show that clusters of iron-rich cells in the rostro-medial upper beak of the pigeon Columbia livia are macrophages, not magnetosensitive neurons. Our systematic characterization of the pigeon upper beak identified iron-rich cells in the stratum laxum of the subepidermis, the basal region of the respiratory epithelium and the apex of feather follicles. Using a three-dimensional blueprint of the pigeon beak created by magnetic resonance imaging and computed tomography, we mapped the location of iron-rich cells, revealing unexpected variation in their distribution and number--an observation that is inconsistent with a role in magnetic sensation. Ultrastructure analysis of these cells, which are not unique to the beak, showed that their subcellular architecture includes ferritin-like granules, siderosomes, haemosiderin and filopodia, characteristics of iron-rich macrophages. Our conclusion that these cells are macrophages and not magnetosensitive neurons is supported by immunohistological studies showing co-localization with the antigen-presenting molecule major histocompatibility complex class II. Our work necessitates a renewed search for the true magnetite-dependent magnetoreceptor in birds.

No evidence for intracellular magnetite in putative vertebrate magnetoreceptors identified by magnetic screening.

The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells. Attempts to find these primary sensors have largely relied on the Prussian Blue stain that labels cells rich in ferric iron. This method has proved problematic as it has led investigators to conflate iron-rich macrophages with magnetoreceptors. An alternative approach developed by Eder et al. [Eder SH, et al. (2012) Proc Natl Acad Sci USA 109(30):12022-12027] is to identify candidate magnetoreceptive cells based on their magnetic moment. Here, we explore the utility of this method by undertaking a screen for magnetic cells in the pigeon. We report the identification of a small number of cells (1 in 476,000) with large magnetic moments (8-106 fAm(2)) from various tissues. The development of single-cell correlative light and electron microscopy (CLEM) coupled with electron energy loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM) permitted subcellular analysis of magnetic cells. This revealed the presence of extracellular structures composed of iron, titanium, and chromium accounting for the magnetic properties of these cells. Application of single-cell CLEM to magnetic cells from the trout failed to identify any intracellular structures consistent with biogenically derived magnetite. Our work illustrates the need for new methods to test the magnetite hypothesis of magnetosensation.

Methylene Blue-Guided Debridement as an Intraoperative Adjunct for the Surgical Treatment of Periprosthetic Joint Infection.

BACKGROUND: Current methods to identify infected tissue in periprosthetic joint infection (PJI) are inadequate. The purpose of this study was (1) to assess methylene blue-guided surgical debridement as a novel technique in PJI using quantitative microbiology and (2) to evaluate clinical success based on eradication of infection and infection-free survival. METHODS: Sixteen total knee arthroplasty patients meeting Musculoskeletal Infection Society criteria for PJI undergoing the first stage of 2-stage exchange arthroplasty were included in this prospective study. Dilute methylene blue (0.1%) was instilled in the knee before debridement, residual dye was removed, and stained tissue was debrided. Paired tissue samples, stained and unstained, were collected from the femur, tibia, and capsule during debridement. Samples were analyzed by neutrophil count, semiquantitative culture, and quantitative polymerase chain reaction (PCR). Clinical success was a secondary outcome. RESULTS: The mean age was 64.0 ± 6.0 years, and follow-up was 24.4 ± 3.5 months. More bacteria were found in methylene blue-stained vs unstained tissue-based on semiquantitative culture (P = .001). PCR for staphylococcal species showed 9-fold greater bioburden in methylene blue-stained vs unstained tissue (P = .02). Tissue pathology found 53 ± 46 polymorphonuclear leukocytes per high-power field in methylene blue-stained vs 4 ± 13 in unstained tissue (P = .0001). All subjects cleared their primary infection and underwent reimplantation. At mean 2-year follow-up, 25% of patients failed secondary to new infection with a different organism. CONCLUSION: These results suggest a role for methylene blue in providing a visual index of surgical debridement in the treatment of PJI.

Labeling of cancer cells with magnetic nanoparticles for magnetic resonance imaging.

PURPOSE: The process of invasion and metastasis formation of tumor cells can be studied by following the migration of labeled cells over prolonged time periods. This report investigates the applicability of iron oxide nanoparticles as a magnetic resonance imaging (MRI) contrast agent for cell labeling. METHODS: γFe2 O3 nanoparticles prepared with direct flame spray pyrolysis are biofunctionalized with poly-l-lysine (PLL). The nanoparticles within the cells were observed with transmission electron microscopy, bright-field microscopy, and magnetorelaxometry. MRI of labeled cells suspended in agarose was used to estimate the detection limit. RESULTS: PLL-coated particles are readily taken up, stored in intracellular clusters, and gradually degraded by the cells. During cell division, the nanoparticle clusters are divided and split between daughter cells. The MRI detection limit was found to be 25 cells/mm(3) for R2*, and 70 cells/mm(3) for R2. The iron specificity, however, was higher for R2 images. Due to the degradation of intracellular γFe2 O3 to paramagnetic iron ions within 13 days, the R1, R2, and R2* contrast gradually decreased over this time period to approximately 50% of its initial value. CONCLUSIONS: These results suggest that PLL-coated γFe2 O3 nanoparticles can be used as an MRI contrast agent for long-term studies of cell migration. Magn Reson Med 71:1896-1905, 2014. © 2013 Wiley Periodicals, Inc.