Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome.

Neurons express various combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. Here, we use epitope-tagged endogenous NR subunits, expansion light-sheet microscopy, and electron microscopy (EM) connectomics to molecularly characterize synapses in Drosophila. We show that in directionally selective motion-sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type-specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determine patterns of synaptic inputs. In support of this model, we identify a transmembrane protein selectively associated with a subset of spatially restricted synapses and demonstrate its requirement for synapse formation through genetic analysis. We propose that mechanisms that regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites.

Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome.

Neurons express different combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. Here we use epitope tagged endogenous NR subunits, expansion light-sheet microscopy, and EM connectomics to molecularly characterize synapses in Drosophila. We show that in directionally selective motion sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determines patterns of synaptic inputs. In support of this model, we identify a transmembrane protein associated selectively with a subset of spatially restricted synapses and demonstrate through genetic analysis its requirement for synapse formation. We propose that mechanisms which regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites.

Designed nanoparticles elicit cross-reactive antibody responses to conserved influenza virus hemagglutinin stem epitopes.

Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that tandem copies of an alpha-helical fragment from the conserved stem region (helix-A) can be displayed on the protruding spikes structures of a capsid scaffold. The stem region of HA on these designed chimeric nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, mice vaccinated with H1-nanoparticles were protected from lethal challenge with H1N1 influenza virus. By using a nanoparticle library approach with this helix-A nanoparticle design, we show that this vaccine nanoparticle construct design could be applicable to different influenza HA subtypes. Importantly, antibodies elicited by H1, H5, and H7 nanoparticles demonstrated homosubtypic and heterosubtypic cross-reactivity binding to different HA subtypes. Also, helix-A nanoparticle immunizations were used to isolate mouse monoclonal antibodies that demonstrated heterosubtypic cross-reactivity and provided protection to mice from viral challenge via passive-transfer. This tandem helix-A nanoparticle construct represents a novel design to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes.

Commercial influenza vaccines vary in HA-complex structure and in induction of cross-reactive HA antibodies.

Influenza virus infects millions of people annually and can cause global pandemics. Hemagglutinin (HA) is the primary component of commercial influenza vaccines (CIV), and antibody titer to HA is a primary correlate of protection. Continual antigenic variation of HA requires that CIVs are reformulated yearly. Structural organization of HA complexes have not previously been correlated with induction of broadly reactive antibodies, yet CIV formulations vary in how HA is organized. Using electron microscopy to study four current CIVs, we find structures including: individual HAs, starfish structures with up to 12 HA molecules, and novel spiked-nanodisc structures that display over 50 HA molecules along the complex's perimeter. CIV containing these spiked nanodiscs elicit the highest levels of heterosubtypic cross-reactive antibodies in female mice. Here, we report that HA structural organization can be an important CIV parameter and can be associated with the induction of cross-reactive antibodies to conserved HA epitopes.

Utility of prolonged prophylactic systemic antibiotics for wound drains in posterior spine surgery: a systematic review and meta-analysis.

OBJECTIVE: Closed suction drains, often used after posterior spinal surgery, pose a potential risk of infection. To combat this risk, many surgeons opt for a prolonged prophylactic antibiotic regimen. Since 2015, several studies have shown that prolonged prophylactic systemic antibiotics (PPSA) for drains provides no additional benefit in reducing surgical site infection (SSI) rates. However, most of these studies lacked sufficient power to make reliable conclusions. To date, there has been no meta-analysis conducted to further investigate this issue. The aim of this study was to investigate whether a regimen of PPSA reduces the incidence of deep SSIs in adult patients with closed suction drains following posterior spinal surgeries. METHODS: The protocol of the current systematic review was registered with PROSPERO. A systematic review of the literature in PubMed (Medline), Europe PMC, Embase, and Cochrane Review databases was conducted for all relevant literature with the keywords "spine," "antibiotics," "surgical site infection," "prophylaxis," and "drain." Retrospective and prospective studies investigating the effectiveness of PPSA in patients 18 years or older who underwent posterior cervical or thoracolumbar surgery and had postoperative wound drains were included. The primary outcome was the odds ratio for deep SSI based on the intervention (PPSA vs non-PPSA). The secondary outcomes were the rates of superficial and overall SSIs. RESULTS: From a total of 2558 titles identified from the search, 7 studies were chosen for final analysis. Three were randomized controlled trials (RCTs), and 4 were retrospective reviews. A total of 2446 patients were analyzed; 1149 received a PPSA regimen and 1297 received a non-PPSA regimen. Deep SSIs occurred in 45 patients (3.9%) and 46 patients (3.5%) in the PPSA and non-PPSA groups, respectively. The odds ratio for deep SSIs in the PPSA group compared with the non-PPSA group was 1.10 (95% CI 0.69-1.74), which was not statistically significant. Additionally, there were no differences in the rates of superficial and overall SSIs. There was a trend toward increased infections with multidrug-resistant bacteria (Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus) in the PPSA group; however, it was not possible to perform a durable statistical analysis because of the small number of reported organisms in the selected publications. CONCLUSIONS: This meta-analysis demonstrates that there is no reduction in rate of deep, superficial, and overall SSIs with prolonged prophylactic antibiotics after posterior spinal surgery involving the use of closed suction drains.

Novel Dual-Lumen Drainage Catheter to Enhance the Active Evacuation of Complex Fluid Collections.

PURPOSE: To compare the performance of a dual-lumen flushable drainage catheter to a conventional catheter for complex fluid collection drainage. METHODS: Two prototype catheters (20- and 28-F) were created by incorporating a customized infusion lumen within the wall of a large-bore conventional drainage catheter, which facilitated simultaneous irrigation of the drainage lumen and the targeted collection via inward- and outward-facing infusion side holes. These were tested against unaltered 20- and 28-F conventional catheters to determine if the injection of a dedicated flush lumen improved rapidity and completeness of gravity drainage. In vitro models were created to simulate serous fluid, purulent/exudative fluid, particulate debris, and acute hematoma. RESULTS: In the purulent model, mean drainage rate was 19.9 ± 8.0 and 9.5±1.4 mL/min for the 20-F prototype and control (P < .001) and 63.9 ± 4.3 and 35.4 ± 3.4 mL/min for the 28-F prototype and control (P = .006), respectively, with complete drainage achieved in all trials. In the particulate model, mean drainage rate was 24.5 ± 9.7 and 12.0 ± 12.5 mL/min for the 28-F prototype and control (P = .003), respectively, with 69.0% versus 41.1% total drainage achieved over 24 minutes (P = .029). In the hematoma model, mean drainage rate was 22.7 ± 4.6 and 4.8 ± 4.3 mL/min for the 28-F prototype and control (P = .022), respectively, with 80.3% versus 20.1% drainage achieved over 15 minutes (P = .003). Particulate and hematoma 20-F prototypes and conventional trials failed due to immediate occlusion. CONCLUSIONS: The proposed dual-lumen drainage catheter with irrigation of a dedicated flush lumen improved evacuation of complex fluid collections in vitro.

Negative-Stain Transmission Electron Microscopy of Molecular Complexes for Image Analysis by 2D Class Averaging.

Negative-stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo-EM image processing have maximized the information gained from averaging large numbers of particles. These developments can now be applied back to negative-stain image analysis to ascertain domain level molecular structure (10 to 20 Å) more quickly and efficiently than possible by atomic resolution cryo-EM. Using uranyl acetate stained molecular complexes of influenza hemagglutinin bound to Fab 441D6, we describe a simple and efficient means to collect several hundred micrographs with SerialEM. Using RELION, we illustrate how tens of thousands of complexes can be auto-picked and classified to accurately describe the domain level topology of this unconventional hemagglutinin head-domain epitope. By comparing to the cryo-EM density map of the same complex, we show that questions about epitope mapping and conformational heterogeneity can readily be answered by this negative-stain method. © 2019 The Authors.

Unique Intradural Inflammatory Mass Containing Precipitated Morphine: Confirmatory Analysis by LESA-MS and MALDI-MS.

Opioids are often used for analgesia via continuous intrathecal delivery by implantable devices. A higher concentration and daily dose of opioid have been postulated as risk factors for intrathecal granuloma formation. We present a 42-year-old female patient with chronic abdominal pain from refractory pancreatitis, with an intrathecal drug delivery device implanted 21 years prior, delivering continuous intrathecal morphine. After many years without concerning physical signs or complaints, with gradual increases in daily morphine dose, she presented with rapidly progressive neurologic deficits, including lower extremity, bladder, and bowel symptoms. These symptoms were determined to be secondary to mass effect and local inflammation related to an intrathecal catheter tip granuloma, detected on magnetic resonance imaging of the spine. The mass was urgently resected. On histopathologic examination, this granuloma was found to be unique, in that in addition to the expected inflammatory components, it appeared to contain precipitated nonpolarizable crystals. These were identified as precipitated morphine using liquid extraction surface analysis-tandem mass spectrometry (LESA-MS/MS) and matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance-mass spectrometry imaging (MALDI-FTICR-MSI). In addition to the unique finding of precipitated morphine crystals, the long-term follow-up of both morphine concentration and daily dose increases provides insight into the formation of intrathecal granulomas.

The Role of Transversus Abdominis Plane Blocks in Enhanced Recovery After Surgery Pathways for Open and Laparoscopic Colorectal Surgery.

INTRODUCTION: The concepts of Enhanced Recovery After Surgery (ERAS®) have steadily increased in usage, with benefits in patient outcomes and hospital length of stay. One important component of successful implementation of ERAS protocol is optimized pain control, via the multimodal approach, which includes neuraxial or regional anesthesia techniques and reduction of opioid use as the primary analgesic. Transversus abdominis plane (TAP) block is one such regional anesthesia technique, and it has been widely studied in abdominal surgery. MATERIALS AND METHODS: We performed an extensive literature search in MEDLINE and PubMed. We review the benefits of TAP blocks for colorectal surgery, both laparoscopic and open. We organize the data by surgery type, by method of TAP block performance, and by a comparison of TAP block to alternative analgesic techniques or to placebo. We examine different endpoints, such as postoperative pain, analgesic use, return of bowel function, and length of stay. RESULTS: The majority of studies examined TAP blocks in the context of laparoscopic colorectal surgery, with many, but not all, demonstrating significantly less use of postoperative opioids in comparison to placebo, wound infiltration, and standard postoperative patient-controlled analgesia with intravenous opioid administration. There is evidence that use of liposomal bupivacaine may be more effective than conventional long-acting local anesthetics. Noninferiority of TAP infusions has been demonstrated, compared with continuous thoracic epidural infusions. CONCLUSION: TAP blocks are easily performed, cost-effective, and an opioid-sparing adjunct for laparoscopic colorectal surgery, with minimal procedure-related morbidity. The evidence is in concordance with several of the goals of ERAS pathways.

Evolution of Cell Size Homeostasis and Growth Rate Diversity during Initial Surface Colonization of Shewanella oneidensis.

Cell size control and homeostasis are fundamental features of bacterial metabolism. Recent work suggests that cells add a constant size between birth and division ("adder" model). However, it is not known how cell size homeostasis is influenced by the existence of heterogeneous microenvironments, such as those during biofilm formation. Shewanella oneidensis MR-1 can use diverse energy sources on a range of surfaces via extracellular electron transport (EET), which can impact growth, metabolism, and size diversity. Here, we track bacterial surface communities at single-cell resolution to show that not only do bacterial motility appendages influence the transition from two- to three-dimensional biofilm growth and control postdivisional cell fates, they strongly impact cell size homeostasis. For every generation, we find that the average growth rate for cells that stay on the surface and continue to divide (nondetaching population) and that for cells that detach before their next division (detaching population) are roughly constant. However, the growth rate distribution is narrow for the nondetaching population, but broad for the detaching population in each generation. Interestingly, the appendage deletion mutants (ΔpilA, ΔmshA-D, Δflg) have significantly broader growth rate distributions than that of the wild type for both detaching and nondetaching populations, which suggests that Shewanella appendages are important for sensing and integrating environmental inputs that contribute to size homeostasis. Moreover, our results suggest multiplexing of appendages for sensing and motility functions contributes to cell size dysregulation. These results can potentially provide a framework for generating metabolic diversity in S. oneidensis populations to optimize EET in heterogeneous environments.

Laughter-induced syncope.

Reported cases of syncope caused directly by laughter are rare. The common scenario described in a few reports involved episodes of fortuitous laughter, sometimes followed by a short prodrome of lightheadedness, facial flushing, and dizziness, followed by an episode of definite syncope. There were no seizure-like movements, automatisms, or bladder or bowel incontinence. After the syncopal episodes that were seconds in length, the patients regained consciousness, and at that point were fully oriented. These episodes could recur in a similar situation with such laughter. Many of these patients subsequently underwent full syncope workups, without elucidating a primary cardiac or neurologic cause. In this review of laughter-induced syncope, we describe a patient of ours who fit these descriptions. This phenomenon is likely a subtype of benign Valsalva-related syncope, with autonomic reflex arcs coming into play that ultimately result in global cerebral hypoperfusion. Besides the Valsalva produced by a great fit of laughter, laughter itself has its own neuroendocrine and vasculature effects that may play a role.

Identification of diaryl ether-based ligands for estrogen-related receptor α as potential antidiabetic agents.

Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of diaryl ether based thiazolidenediones, which function as selective ligands against this receptor. Series optimization provided several potent analogues that inhibit the recruitment of a coactivator peptide fragment in in vitro biochemical assays (IC(50) < 150 nM) and cellular two-hybrid reporter assays against the ligand binding domain (IC(50) = 1-5 µM). A cocrystal structure of the ligand-binding domain of ERRα with lead compound 29 revealed the presence of a covalent interaction between the protein and ligand, which has been shown to be reversible. In diet-induced murine models of obesity and in an overt diabetic rat model, oral administration of 29 normalized insulin and circulating triglyceride levels, improved insulin sensitivity, and was body weight neutral. This provides the first demonstration of functional activities of an ERRα ligand in metabolic animal models.