Structural basis of MsbA-mediated lipopolysaccharide transport.

Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

Toll-like Receptor 4-Independent Effects of Lipopolysaccharide Identified Using Longitudinal Serum Proteomics.

Sepsis remains one of the most lethal and costly conditions treated in U.S. hospitals, with approximately 50% of cases caused by Gram-negative bacterial infections. Septic shock is induced when lipopolysaccharide (LPS), the main component of Gram-negative outer bacterial membrane, signals through the Toll-like receptor 4 (TLR4) complex. Lethal endotoxemia, a model for septic shock, was induced in WT C57BL6 and TLR4-/- mice by administration of Escherichia coli LPS. WT LPS treated mice showed high morbidity, while PBS treated LPS and treated TLR4-/- mice did not. ANOVA analysis of label-free quantification of longitudinal serum proteome revealed 182 out of 324 proteins in LPS injected WT mice that were significantly changed across four time points (0, 6, 12, and 18 h). No significant changes were identified in the two control groups. From the 182 identified proteins, examples of known sepsis biomarkers were validated by ELISA, which showed similar trends as MS proteomics data. Longitudinal analysis within individual mice produced 3-fold more significantly changed proteins than pair-wise comparison. A subsequent global analysis of WT and TLR4-/- mice identified pathways activated independent of TLR4. These pathways represent possible compensatory mechanisms that allow for control of Gram-negative bacterial infection regardless of host immune status.

Does Robotic-assisted TKA Result in Better Outcome Scores or Long-Term Survivorship Than Conventional TKA? A Randomized, Controlled Trial.

BACKGROUND: Robotic-assisted TKA was introduced to enhance the precision of bone preparation and component alignment with the goal of improving the clinical results and survivorship of TKA. Although numerous reports suggest that bone preparation and knee component alignment may be improved using robotic assistance, no long-term randomized trials of robotic-assisted TKA have shown whether this results in improved clinical function or survivorship of the TKA. QUESTIONS/PURPOSES: In this randomized trial, we compared robotic-assisted TKA to manual-alignment techniques at long-term follow-up in terms of (1) functional results based on Knee Society, WOMAC, and UCLA Activity scores; (2) numerous radiographic parameters, including component and limb alignment; (3) Kaplan-Meier survivorship; and (4) complications specific to robotic-assistance, including pin-tract infection, peroneal nerve palsy, pin-site fracture, or patellar complications. METHODS: This study was a registered prospective, randomized, controlled trial. From January 2002 to February 2008, one surgeon performed 975 robotic-assisted TKAs in 850 patients and 990 conventional TKAs in 849 patients. Among these patients 1406 patients were eligible for participation in this study based on prespecified inclusion criteria. Of those, 100% (1406) patients agreed to participate and were randomized, with 700 patients (750 knees) receiving robotic-assisted TKA and 706 patients (766 knees) receiving conventional TKA. Of those, 96% (674 patients) in the robotic-assisted TKA group and 95% (674 patients) in the conventional TKA group were available for follow-up at a mean of 13 (± 5) years. In both groups, no patient older than 65 years was randomized because we anticipated long-term follow-up. We evaluated 674 patients (724 knees) in each group for clinical and radiographic outcomes, and we examined Kaplan-Meier survivorship for the endpoint of aseptic loosening or revision. Clinical evaluation was performed using the original Knee Society knee score, the WOMAC score, and the UCLA activity score preoperatively and at latest follow-up visit. We also assessed loosening (defined as change in the position of the components) using plain radiographs, osteolysis using CT scans at the latest follow-up visit, and component, and limb alignment on mechanical axis radiographs. To minimize the chance of type-2 error and increase the power of our study, we assumed the difference in the Knee Society score to be 25 points to match the MCID of the Knee Society score with a SD of 5; to be able to detect a difference of this size, we calculated that a total of 628 patients would be needed in each group in order to achieve 80% power at the α = 0.05 level. RESULTS: Clinical parameters at the latest follow-up including the Knee Society knee scores (93 ± 5 points in the robotic-assisted TKA group versus 92 ± 6 points in the conventional TKA group [95% confidence interval 90 to 98]; p = 0.321) and Knee Society knee function scores (83 ± 7 points in the robotic-assisted TKA group versus 85 ± 6 points in the conventional TKA group [95% CI 75 to 88]; p = 0.992), WOMAC scores (18 ± 14 points in the robotic-assisted TKA group versus 19 ± 15 points in the conventional TKA group [95% CI 16 to 22]; p = 0.981), range of knee motion (125 ± 6° in the robotic-assisted TKA group versus 128 ± 7° in the conventional TKA group [95% CI 121 to 135]; p = 0.321), and UCLA patient activity scores (7 points versus 7 points in each group [95% CI 5 to 10]; p = 1.000) were not different between the two groups at a mean of 13 years' follow-up. Radiographic parameters such as the femorotibial angle (mean 2° ± 2° valgus in the robotic-assisted TKA group versus 3° ± 3° valgus in the conventional TKA group [95% CI 1 to 5]; p = 0.897), femoral component position (coronal plane: mean 98° in the robotic-assisted TKA group versus 97° in the conventional TKA group [95% CI 96 to 99]; p = 0.953; sagittal plane: mean 3° in the robotic-assisted TKA group versus 2° in the conventional TKA group [95% CI 1 to 4]; p = 0.612) and tibial component position (coronal plane: mean 90° in the robotic-assisted TKA group versus 89° in the conventional TKA group [95% CI 87 to 92]; p = 0.721; sagittal plane: 87° in the robotic-assisted TKA group versus 86° in the conventional TKA group [95% CI 84 to 89]; p = 0.792), joint line (16 mm in the robotic-assisted TKA group versus 16 mm in the conventional TKA group [95% CI 14 to 18]; p = 0.512), and posterior femoral condylar offset (24 mm in the robotic-assisted TKA group versus 24 mm in the conventional TKA group [95% CI 21 to 27 ]; p = 0.817) also were not different between the two groups (p > 0.05). The aseptic loosening rate was 2% in each group, and this was not different between the two groups. With the endpoint of revision or aseptic loosening of the components, Kaplan-Meier survivorship of the TKA components was 98% in both groups (95% CI 94 to 100) at 15 years (p = 0.972). There were no between-group differences in terms of the frequency with which complications occurred. In all, 0.6% of knees (four) in each group had a superficial infection, and they were treated with intravenous antibiotics for 2 weeks [corrected]. No deep infection occurred in these knees. In the conventional TKA group, 0.6% of knees (four) had motion limitation (< 60°) [corrected]. CONCLUSIONS: At a minimum follow-up of 10 years, we found no differences between robotic-assisted TKA and conventional TKA in terms of functional outcome scores, aseptic loosening, overall survivorship, and complications. Considering the additional time and expense associated with robotic-assisted TKA, we cannot recommend its widespread use. LEVEL OF EVIDENCE: Level I, therapeutic study.

Lipopolysaccharides from Different Burkholderia Species with Different Lipid A Structures Induce Toll-Like Receptor 4 Activation and React with Melioidosis Patient Sera.

Lipopolysaccharides (LPSs) of Gram-negative bacteria comprise lipid A, core, and O-polysaccharide (OPS) components. Studies have demonstrated that LPSs isolated from the pathogenic species Burkholderia pseudomallei and Burkholderia mallei and from less-pathogenic species, such as Burkholderia thailandensis, are potent immune stimulators. The LPS structure of B. pseudomallei, the causative agent of melioidosis, is highly conserved in isolates from Thailand; however, the LPSs isolated from other, related species have not been characterized to enable understanding of their immune recognition and antigenicities. Here, we describe the structural and immunological characteristics of the LPSs isolated from eight Burkholderia species and compare those for B. pseudomallei to those for the other seven species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), gas chromatography (GC), SDS-PAGE, Toll-like receptor 4 (TLR4) stimulation, and immunoblot analysis were performed on these Burkholderia species. MALDI-TOF profiles demonstrated that Burkholderia lipid A contains predominantly penta-acylated species modified with 4-amino-4-deoxy-arabinose residues at both terminal phosphate groups. The lipid A could be differentiated based on mass differences at m/z 1,511, 1,642, 1,773, and 1,926 and on fatty acid composition. LPSs of all species induced TLR4-dependent NF-κB responses; however, while SDS-PAGE analysis showed similar LPS ladder patterns for B. pseudomallei, B. thailandensis, and B. mallei, these patterns differed from those of other Burkholderia species. Interestingly, immunoblot analysis demonstrated that melioidosis patient sera cross-reacted with OPSs of other Burkholderia species. These findings can be used to better understand the characteristics of LPS in Burkholderia species, and they have implications for serological diagnostics based on the detection of antibodies to OPS.

Rapid Microbial Identification and Antibiotic Resistance Detection by Mass Spectrometric Analysis of Membrane Lipids.

Infectious diseases have a substantial global health impact. Clinicians need rapid and accurate diagnoses of infections to direct patient treatment and improve antibiotic stewardship. Current technologies employed in routine diagnostics are based on bacterial culture followed by morphological trait differentiation and biochemical testing, which can be time-consuming and labor-intensive. With advances in mass spectrometry (MS) for clinical diagnostics, the U.S. Food and Drug Administration has approved two microbial identification platforms based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of microbial proteins. We recently reported a novel and complementary approach by comparing MALDI-TOF mass spectra of microbial membrane lipid fingerprints to identify ESKAPE pathogens. However, this lipid-based approach used a sample preparation method that required more than a working day from sample collection to identification. Here, we report a new method that extracts lipids efficiently and rapidly from microbial membranes using an aqueous sodium acetate (SA) buffer that can be used to identify clinically relevant Gram-positive and -negative pathogens and fungal species in less than an hour. The SA method also has the ability to differentiate antibiotic-susceptible and antibiotic-resistant strains, directly identify microbes from biological specimens, and detect multiple pathogens in a mixed sample. These results should have positive implications for the manner in which bacteria and fungi are identified in general hospital settings and intensive care units.

Fluoroscopic Balloon Dilation Using a Flexible Guide Wire to Treat Obstructive Eustachian Tube Dysfunction.

PURPOSE: To prospectively evaluate the midterm outcomes of fluoroscopic eustachian tube (E-tube) balloon dilation by using a flexible guide wire in patients with obstructive E-tube dysfunction. MATERIALS AND METHODS: From October 2016 to September 2017, adult outpatients with persistent otitis media who were unable to perform the Valsalva maneuver were prospectively enrolled. The analysis included 32 E-tubes from 31 patients (18 women, 13 men; mean age, 47 years old, range 25-72 years). Participants underwent fluoroscopic E-tube balloon dilation with a 0.035-inch flexible guide wire and a 6- × 20-mm balloon catheter. Clinical examinations to check for the ability to perform the Valsalva maneuver and otomicroscopy were conducted at 1 week and then at 1, 3, 12, and 18 months after the procedure. RESULTS: Balloon dilation was technically successful in all E-tubes. The mean time required for the procedure was 6.9 minutes (range, 5.8-10.3 minutes). The Valsalva maneuver was successful in opening 25 of 32 E-tubes (78.1%) at 3 months after balloon dilation. During the median follow-up of 15.9 months, failure of the Valsalva maneuver occurred in 4 of 25 improved E-tubes (16%), yielding a 2-year patency rate of 84%. CONCLUSIONS: The fluoroscopic balloon dilation results were encouraging, and using a flexible guide wire for E-tube balloon dilation did not cause a false passage.

Structural modification of LPS in colistin-resistant, KPC-producing Klebsiella pneumoniae.

BACKGROUND: Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited. OBJECTIVES: To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains. METHODS: The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS. RESULTS: All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype. CONCLUSIONS: The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings.

EW-7197, an activin-like kinase 5 inhibitor, suppresses granulation tissue after stent placement in rat esophagus.

BACKGROUND AND AIMS: Self-expanding metallic stent (SEMS) placement is a well-established method for treating malignant esophageal strictures; however, this procedure has not gained widespread acceptance for treating benign esophageal strictures because of granulation tissue formation. The aim of the present study was to investigate whether EW-7197, a novel per-oral transforming growth factor-ß type I receptor kinase inhibitor, suppressed granulation tissue formation after SEMS placement in the rat esophagus. METHODS: Sixty rats underwent SEMS placement and were randomly divided into 4 groups. Group A (n = 20) received vehicle-treated control for 4 weeks. Group B (n = 20) received 20 mg/kg/day EW-7197 for 4 weeks. Group C (n = 10) received 20 mg/kg/day EW-7197 for 4 weeks followed by vehicle-treated control for 4 weeks. Group D (n = 10) received 20 mg/kg/day EW-7197 for 8 weeks. RESULTS: SEMS placement was technically successful in all rats. Eleven rats, however, were excluded because of stent migration (n = 9) and procedure-related death (n = 2). The luminal diameter in group A was significantly smaller than those in groups B, C, and D (all P < .001). The percentage of granulation tissue area, number of epithelial layers, thickness of submucosal fibrosis, percentage of connective tissue area, and degree of collagen deposition were significantly higher in group A than in groups B, C, and D (all P < .001); however, there were no significant differences among groups B, C, and D. EW-7197 decreased the expression levels of phospho-Smad 3, N-cadherin, fibronectin, α-smooth muscle actin, and transforming growth factor-ß1 and increased the expression level of E-cadherin (all P < .01). CONCLUSIONS: EW-7197 suppressed granulation tissue formation after SEMS placement in the rat esophagus.

Fluoroscopic Balloon Dilation for Treating Postoperative Nonanastomotic Strictures in the Proximal Small Bowel: A 15-Year Single-Institution Experience.

PURPOSE: To evaluate safety and effectiveness of fluoroscopic balloon dilation (FBD) for treating postoperative nonanastomotic strictures in proximal small bowel. MATERIALS AND METHODS: Data of 44 patients (26 men and 18 women; mean age, 53.7 y ± 13.0) treated with FBD for postoperative nonanastomotic strictures in the proximal small bowel between January 2000 and February 2016 were retrospectively reviewed. Site of stricture was located in the first portion of duodenum in 8 (18.2%) patients, second portion of duodenum in 8 (18.2%) patients, third portion of duodenum in 1 (2.3%) patient, fourth portion of duodenum in 1 (2.3%) patient, and proximal jejunum in 26 (59.1%) patients. Mean distance between the most anal-side lesion and the oral cavity was 63.9 cm ± 15.0. RESULTS: Technical success was achieved in 39 (88.6%) patients. Mean stricture length was 3.0 cm ± 1.8. Technical failure because of inability to negotiate the guide wire through the stricture occurred in 5 (13.6%) patients. Complete resolution of obstructive symptoms and resumption of oral intake of soft or solid food within 3 days occurred in 34 patients after 1 (n = 32) or 2 (n = 2) FBD sessions, rendering a clinical success rate of 87.2%. There were no major complications directly related to FBD. Median follow-up period was 1,406 days (interquartile range, 594-2,236 d). Nine (26.5%) patients had recurrence within a median 47 days (interquartile range, 20-212 d). CONCLUSIONS: FBD may be safe and effective for treating postoperative nonanastomotic strictures in the proximal small bowel.

Structural derivation of lipid A from Cronobacter sakazakii using tandem mass spectrometry.

RATIONALE: Cronobacter sakazakii is a Gram-negative opportunistic pathogen that can cause necrotizing enterocolitis, bacteremia, and meningitis. Lipid A, the glycolipid membrane anchor of lipopolysaccharide (LPS), is a potential virulence factor for C. sakazakii. Given the potential importance of this molecule in infection and virulence, structural characterization of lipid A was carried out. METHODS: The structural characterization of lipid A extracted from C. sakazakii was performed using electrospray ionization and collision-induced dissociation in a linear ion trap mass spectrometer. Specifically, for detailed structural characterization, hierarchical tandem mass spectrometry was performed on the dominant ions present in the precursor ion mass spectra. By comparing the C. sakazakii fragmentation pathways to those of the known structure of E. coli lipid A, a structure of C. sakazakii lipid A was derived. RESULTS: The precursor ion at m/z 1796 from C. sakazakii is produced from a lipid A molecule where the acyl chains between the 2'b (C14) and 3'b (C12) positions are reversed as compared to E. coli lipid A. Additionally, the precursor ion at m/z 1824 from C. sakazakii corresponds to an E. coli structure with the same acyl chain at the 2'b position (C14), but a longer acyl chain (C14) at the 3'b position versus m/z 1796. CONCLUSIONS: Two lipid A structures were derived for the C. sakazakii ions at m/z 1796 and 1824. They differed in composition at the 2'b and 3'b acyl chain substituents, which may be a result of differences in substrate specificity of the two lipid A acyl chain transferases: LpxL and LpxM. Copyright © 2016 John Wiley & Sons, Ltd.

Rapid lipid a structure determination via surface acoustic wave nebulization and hierarchical tandem mass spectrometry algorithm.

RATIONALE: Surface acoustic wave nebulization (SAWN) is an easy to use sample transfer method for rapid mass spectrometric analysis. A new standing wave (SW) SAWN chip, with higher ionization efficiency than our previously reported design, is used for rapid analysis of lipids. METHODS: The crude, yet fast, Caroff protocol was used for lipid A extraction from Francisella novicida. SW-SAWN with a Waters Synapt G2S quadrupole time-of-flight (QTOF) mass spectrometer was used to generate lipid A ions. Quadrupole collision-induced dissociation (Q-CID) of lipid A at varying CID energies was used to approximate the ion trap MSn data required for our hierarchical tandem mass spectrometry (HiTMS) algorithm. Structural hypotheses can be obtained directly from the HiTMS algorithm to identify species-specific lipid A molecules. RESULTS: SW-SAWN successfully generated ions from lipid A extracted from Francisella novicida using the faster Caroff method. In addition, varying collision energies were used to generate tandem mass spectra similar to MS3 and MS4 spectra from an ion trap. The Q-CID spectra are compatible with our HiTMS algorithm and offer an improvement over lipid A tandem mass spectra acquired in an ion trap. CONCLUSIONS: Combining SW-SAWN and Q-CID enabled more structural assignments than previously reported in half the time. The ease of generating spectra by SAWN tandem MS in combination with HiTMS interpretation offers high-throughput lipid A structural analysis and thereby rapid detection of pathogens based on lipid fingerprinting. Copyright © 2016 John Wiley & Sons, Ltd.

Methemoglobin is an endogenous toll-like receptor 4 ligand-relevance to subarachnoid hemorrhage.

Neuroinflammation is a well-recognized consequence of subarachnoid hemorrhage (SAH), and may be responsible for important complications of SAH. Signaling by Toll-like receptor 4 (TLR4)-mediated nuclear factor κB (NFκB) in microglia plays a critical role in neuronal damage after SAH. Three molecules derived from erythrocyte breakdown have been postulated to be endogenous TLR4 ligands: methemoglobin (metHgb), heme and hemin. However, poor water solubility of heme and hemin, and lipopolysaccharide (LPS) contamination have confounded our understanding of these molecules as endogenous TLR4 ligands. We used a 5-step process to obtain highly purified LPS-free metHgb, as confirmed by Fourier Transform Ion Cyclotron Resonance mass spectrometry and by the Limulus amebocyte lysate assay. Using this preparation, we show that metHgb is a TLR4 ligand at physiologically relevant concentrations. metHgb caused time- and dose-dependent secretion of the proinflammatory cytokine, tumor necrosis factor α (TNFα), from microglial and macrophage cell lines, with secretion inhibited by siRNA directed against TLR4, by the TLR4-specific inhibitors, Rs-LPS and TAK-242, and by anti-CD14 antibodies. Injection of purified LPS-free metHgb into the rat subarachnoid space induced microglial activation and TNFα upregulation. Together, our findings support the hypothesis that, following SAH, metHgb in the subarachnoid space can promote widespread TLR4-mediated neuroinflammation.

Identification and characterization of a drug-sensitive strain enables puromycin-based translational assays in Saccharomyces cerevisiae.

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C-terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6∆, pdr1∆ and pdr3∆ (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin-based assays in yeast, which will enable new avenues of inquiry into protein production and maturation.

Transvaginal specimen extraction versus conventional minilaparotomy after laparoscopic anterior resection for colorectal cancer: mid-term results of a case-matched study.

BACKGROUND: Although the vagina is considered a viable route during laparoscopic surgery, a number of concerns have led to a need to demonstrate the safety of a transvaginal approach in colorectal surgery. However, the data for transvaginal access in left-sided colorectal cancer are extremely limited, and no study has compared the clinical outcomes with a conventional laparoscopic procedure. OBJECTIVE: We compared the clinical outcomes of totally laparoscopic anterior resection with transvaginal specimen extraction (TVSE) with those of the conventional laparoscopic approach with minilaparotomy (LAP) for anastomosis construction and specimen retrieval in left-sided colorectal cancer. METHODS: Fifty-eight patients underwent TVSE between October 2006 and July 2011 and were matched by age, surgery date, tumor location, and tumor stage with patients who underwent conventional LAP for left-sided colorectal cancer. RESULTS: Operative time was significantly longer in the TVSE group (149.3 ± 39.8 vs. 131.9 ± 41.4 min; p = 0.023). Patients in the TVSE group experienced less pain (pain score 4.9 ± 1.6 vs. 5.8 ± 1.9; p = 0.008), shorter time to passage of flatus (2.2 ± 1.1 vs. 2.7 ± 1.2 days; p = 0.026), and higher satisfaction with the cosmetic results (cosmetic score 8.0 ± 1.4 vs. 6.3 ± 1.5; p = 0.001). More endolinear staplers for rectal transection were used in the LAP group (1.2 ± 0.5 vs. 1.1 ± 0.2; p = 0.021). Overall morbidities were similar in both groups; however, three wound infections only occurred in the LAP group. After a median follow-up of 34.4 (range 11-60) months, no transvaginal access-site recurrence occurred. The 3-year disease-free survival was similar between groups (91.5 vs. 90.8%; p = 0.746). CONCLUSIONS: Transvaginal access after totally laparoscopic anterior resection is safe and feasible for left-sided colorectal cancer in selected patients with better short-term outcomes.

Myristoylated, Alanine-rich C-kinase Substrate (MARCKS) regulates Toll-like receptor 4 signaling in macrophages.

MARCKS (Myristoylated Alanine-rich C-kinase Substrate) is a membrane protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (Toll-like receptor 4). The presence of MARCKS and the formation of phospho-MARCKS in macrophages have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. To investigate the role of MARCKS during inflammation, we activated macrophages using LPS with or without the addition of a PKC inhibitor. We found that PKC inhibition substantially decreased macrophage IL6 and TNF cytokine production. In addition, confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation. CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated TNF and IL6 production, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes and signaling pathways, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory related diseases.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) regulates toll-like receptor 4 signaling in macrophages.

MARCKS (myristoylated alanine-rich C-kinase substrate) is a membrane-associated protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (toll-like receptor 4).The presence of MARCKS and the formation of phospho-MARCKS in various cell types have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. We investigated the role of MARCKS in inflammation. Confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation.CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated the production of TNF and IL6, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes including innate immune response, inflammatory response, cytokine production, and molecular functions such as extracellularly ATP-gated cation channel activity, electron transfer activity and oxidoreductase activity, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory diseases.

The minichromosome maintenance complex drives esophageal basal zone hyperplasia.

Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen-driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography-tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell-related proteins were among the most highly upregulated DEPs in EoE compared with controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all 6 subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of epithelial cells through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.

The rat eustachian tube: Anatomical, histological, and radiological features.

Purpose: This study investigated the anatomical and histological characteristics of the rat Eustachian tube (E-tube) and the feasibility of Eustachian tubography in a rat model. Materials and methods: Fifteen male Wistar rats were used in this study, and the bilateral E-tubes of each rat were examined. Ten E-tubes were used for anatomical studies, another ten for histological analysis, and the other ten for Eustachian tubography. Five rats were euthanized and decapitated, and ten E-tubes were dissected to describe the anatomy of the E-tube. Ten E-tube specimens obtained from five other rats were sectioned to investigate E-tube histology. Eustachian tubography was performed on the bilateral E-tubes of the other five rats using the trans-tympanic approach. Results: The rat E-tubes consisted of bony and membranous parts. Cartilage and bone tissue covered only the bony part. The E-tubes' mean diameter and overall length were 2.97 â€‹mm and 4.96 â€‹mm, respectively. The tympanic orifices' mean diameter was 1.21 â€‹mm. The epithelium of E-tubes was mainly composed of pseudostratified ciliated and goblet cells. Eustachian tubography was successfully performed on both sides of the E-tube for each rat. The technical success rate was 100%, the average running time was 4.9 â€‹min, and no procedure-related complications occurred. On tubography images, the E-tube, tympanic cavity, and nasopharynx could be identified because of the visualization of bony landmarks. Conclusion: In this study, we described the anatomical and histological features of rat E-tubes. With the aid of these findings, E-tube angiography was successfully performed using a transtympanic approach. These results will facilitate further investigation of E-tube dysfunction.