Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix.

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.

Evolution avoids a pathological stabilizing interaction in the immune protein S100A9.

Stability constrains evolution. While much is known about constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently identified a mutation in the innate immune protein S100A9 that provides insight into such constraints. When introduced into human S100A9, M63F simultaneously increases the stability of the protein and disrupts its natural ability to activate Toll-like receptor 4. Using chemical denaturation, we found that M63F stabilizes a calcium-bound conformation of hS100A9. We then used NMR to solve the structure of the mutant protein, revealing that the mutation distorts the hydrophobic binding surface of hS100A9, explaining its deleterious effect on function. Hydrogen-deuterium exchange (HDX) experiments revealed stabilization of the region around M63F in the structure, notably Phe37. In the structure of the M63F mutant, the Phe37 and Phe63 sidechains are in contact, plausibly forming an edge-face π-stack. Mutating Phe37 to Leu abolished the stabilizing effect of M63F as probed by both chemical denaturation and HDX. It also restored the biological activity of S100A9 disrupted by M63F. These findings reveal that Phe63 creates a molecular staple with Phe37 that stabilizes a nonfunctional conformation of the protein, thus disrupting function. Using a bioinformatic analysis, we found that S100A9 proteins from different organisms rarely have Phe at both positions 37 and 63, suggesting that avoiding a pathological stabilizing interaction indeed constrains S100A9 evolution. This work highlights an important evolutionary constraint on stabilizing mutations, namely, that they must avoid inappropriately stabilizing nonfunctional protein conformations.

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions.

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.

Hydrothermal Alkaline Treatment (HALT) of Foam Fractionation Concentrate Derived from PFAS-Contaminated Groundwater.

While foam fractionation (FF) process has emerged as a promising technology for removal of per- and polyfluoroalkyl substances (PFASs) from contaminated groundwater, management of the resulting foam concentrates with elevated concentrations of PFASs (e.g., >1 g/L) remains a challenge. Here, we applied hydrothermal alkaline treatment (HALT) to two foam concentrates derived from FF field demonstration projects that treated aqueous film-forming foam (AFFF)-impacted groundwater. Results showed >90% degradation and defluorination within 90 min of treatment (350 °C, 1 M NaOH) of all 62 PFASs (including cations, anions, and zwitterions) identified in foam concentrates. Observed rate constants for degradation of individual perfluoroalkyl sulfonates (PFSAs, CnF2n+1-SO3-), the most recalcitrant class of PFASs, in both foam concentrates were similar to values measured previously in other aqueous matrices, indicating that elevated initial PFAS concentrations (e.g., PFHxSinit = 0.55 g/L), dissolved organic carbon (DOC; up to 4.5 g/L), and salt levels (e.g., up to 325 mg/L chloride) do not significantly affect PFAS reaction kinetics. DOC was partially mineralized by treatment, but a fraction (∼15%) was recalcitrant. Spectroscopic characterization revealed molecular features of the HALT-recalcitrant DOC fraction, and nontarget high-resolution mass spectrometry tentatively identified 129 nonfluorinated HALT-recalcitrant molecules. Analysis of process energy requirements shows that treating PFAS-contaminated foam concentrates with HALT would add minimally (<5%) to the overall energy requirements of an integrated FF-HALT treatment train.

Multivalent binding of the hub protein LC8 at a newly discovered site in 53BP1.

Tumor suppressor p53 binding protein 1 (53BP1) is a scaffolding protein involved in poly-ADP ribose polymerase inhibitor hypersensitivity in BRCA1-negative cancers. 53BP1 plays a critical role in the DNA damage response and relies on its oligomerization to create foci that promote repair of DNA double-strand breaks. Previous work shows that mutation of either the oligomerization domain or the dynein light chain 8 (LC8)-binding sites of 53BP1 results in reduced accumulation of 53BP1 at double-strand breaks. Mutation of both abolishes focus formation almost completely. Here, we show that, contrary to current literature, 53BP1 contains three LC8-binding sites, all of which are conserved in mammals. Isothermal titration calorimetry measuring binding affinity of 53BP1 variants with LC8 shows that the third LC8-binding site has a high affinity and can bind LC8 in the absence of other sites. NMR titrations confirm that the third site binds LC8 even in variants that lack the other LC8-binding sites. The third site is the closest to the oligomerization domain of 53BP1, and its discovery would challenge our current understanding of the role of LC8 in 53BP1 function.

Paints: A Source of Volatile PFAS in Air─Potential Implications for Inhalation Exposure.

Paints are widely used in indoor settings yet there are no data for volatile per- and polyfluoroalkyl substances (PFAS) for paints or knowledge if paints are potentially important sources of human exposure to PFAS. Different commercial paints (n = 27) were collected from local hardware stores and analyzed for volatile PFAS by gas chromatography-mass spectrometry (GC-MS), nonvolatile PFAS by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF), and total fluorine by 19F nuclear magnetic resonance spectroscopy (NMR). Diluted paint required clean up to remove 6:2 fluorotelomer phosphate diester (diPAP), which thermally transforms into 6:2 FTOH at 280 °C (GC inlet temperature). Only 6:2 FTOH (0.9-83 µg/g) and 6:2 diPAP (0.073-58 µg/g) were found in five exterior and nine interior paints and only accounted for a maximum of 17% of total fluorine. Upon drying, 40% of the FTOH mass was lost, and the loss was verified by measurements of the cumulative FTOH mass measured in the air of a small, confined space over a 3 h period. Based on the liquid paint results, the ConsExpo model was used for potential exposure assessment and one commercial paint exceeded the chosen reference dose (5 µg/kg-day) for children and adults, indicating the potential for human exposure during painting.

Postoperative bleeding and leaks in sleeve gastrectomy are independent of both staple height and staple line oversewing.

BACKGROUND: Over 100,000 sleeve gastrectomy procedures are performed annually in the USA. Despite technological advances, postoperative bleeding and gastric staple line leak are complications of this procedure. We analyzed patient-specific and perioperative factors to determine their association with these complications. METHODS: We performed a retrospective cohort analysis of patients who underwent sleeve gastrectomy between 2005 and 2019 at our institution. Patient demographics, comorbidities, and procedure details including type of energy device, staple type, staple height, staple line oversewing, and staple line clipping were compared using multiple logistic regression for combined postoperative complications (blood transfusion, bleeding, and staple line leak). Postoperative bleeding was defined by requiring blood transfusion and/or re-operation to control bleeding. Staple line leak was confirmed radiographically. RESULTS: There were 1213 patients who underwent sleeve gastrectomy. Fifty-two high-risk patients were excluded due to cirrhosis, end-stage renal disease, and anticoagulation use for left ventricular assist device. Of the remaining 1161 patients, twenty-five (2.2%) received postoperative blood transfusion, nine (0.8%) had postoperative bleeding, two (0.2%) had staple line leak, and twenty-eight patients (2.4%) had combined postoperative complications. The median age was significantly higher for patients with combined postoperative complications (43 vs 49; p = 0.02). There was no difference in postoperative blood transfusion, bleeding, staple line leak, or combined postoperative complication with different energy devices (p = 0.92), staple types (p = 0.21), staple heights (p = 0.50), or staple line suturing/clipping (p = 0.95). In addition, there was no difference in bleeding when comparing staple line sewing techniques (p = 0.44). Predictably, patients with combined postoperative complications had increased length of stay (3 days vs 1 day; p < 0.001). CONCLUSION: Sleeve gastrectomy procedure has tremendous variability in technique and devices used. We observed no difference in the combined postoperative complications of bleeding or staple line leak with respect to different energy devices, staple height, or oversewing of the gastric staple line. Patient selection is crucial, as patient age and coagulopathic comorbidities were found to lead to higher combined postoperative complications.

Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA.

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1-1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1-1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.

The dynein light chain 8 (LC8) binds predominantly "in-register" to a multivalent intrinsically disordered partner.

Dynein light chain 8 (LC8) interacts with intrinsically disordered proteins (IDPs) and influences a wide range of biological processes. It is becoming apparent that among the numerous IDPs that interact with LC8, many contain multiple LC8-binding sites. Although it is established that LC8 forms parallel IDP duplexes with some partners, such as nucleoporin Nup159 and dynein intermediate chain, the molecular details of these interactions and LC8's interactions with other diverse partners remain largely uncharacterized. LC8 dimers could bind in either a paired "in-register" or a heterogeneous off-register manner to any of the available sites on a multivalent partner. Here, using NMR chemical shift perturbation, analytical ultracentrifugation, and native electrospray ionization MS, we show that LC8 forms a predominantly in-register complex when bound to an IDP domain of the multivalent regulatory protein ASCIZ. Using saturation transfer difference NMR, we demonstrate that at substoichiometric LC8 concentrations, the IDP domain preferentially binds to one of the three LC8 recognition motifs. Further, the differential dynamic behavior for the three sites and the size of the fully bound complex confirmed an in-register complex. Dynamics measurements also revealed that coupling between sites depends on the linker length separating these sites. These results identify linker length and motif specificity as drivers of in-register binding in the multivalent LC8-IDP complex assembly and the degree of compositional and conformational heterogeneity as a promising emerging mechanism for tuning of binding and regulation.

Magnetic Sphincter Augmentation Superior to Proton Pump Inhibitors for Regurgitation in a 1-Year Randomized Trial.

BACKGROUND & AIMS: Regurgitative gastroesophageal reflux disease (GERD) refractive to medical treatment is common and caused by mechanical failure of the anti-reflux barrier. We compared the effects of magnetic sphincter augmentation (MSA) with those of proton-pump inhibitors (PPIs) in a randomized trial. METHODS: Patients with moderate to severe regurgitation (assessed by the foregut symptom questionnaire) despite once-daily PPI therapy (n = 152) were randomly assigned to groups given twice-daily PPIs (n = 102) or laparoscopic MSA (n = 50) at 20 sites, from July 2015 through February 2017. Patients answered questions from the foregut-specific reflux disease questionnaire and GERD health-related quality of life survey about regurgitation, heartburn, dysphagia, bloating, diarrhea, flatulence, and medication use, at baseline and 6 and 12 months after treatment. Six months after PPI therapy, MSA was offered to patients with persistent moderate to severe regurgitation and excess reflux episodes during impedance or pH testing on medication. Regurgitation, foregut scores, esophageal acid exposure, and adverse events were evaluated at 1 year. RESULTS: Patients in the MSA group and those who crossed over to the MSA group after PPI therapy (n = 75) had similar outcomes. MSA resulted in control of regurgitation in 72/75 patients (96%); regurgitation control was independent of preoperative response to PPIs. Only 8/43 patients receiving PPIs (19%) reported control of regurgitation. Among the 75 patients who received MSA, 61 (81%) had improvements in GERD health-related quality of life improvement scores (greater than 50%) and 68 patients (91%) discontinued daily PPI use. Proportions of patients with dysphagia decreased from 15% to 7% (P < .005), bloating decreased from 55% to 25%, and esophageal acid exposure time decreased from 10.7% to 1.3% (P < .001) from study entry to 1-year after MSA (Combined P < .001). Seventy percent (48/69) of patients had pH normalization at study completion. MSA was not associated with any peri-operative events, device explants, erosions, or migrations. CONCLUSIONS: In a prospective study, we found MSA to reduce regurgitation in 95% of patients with moderate to severe regurgitation despite once-daily PPI therapy. MSA is superior to twice-daily PPIs therapy in reducing regurgitation. Relief of regurgitation is sustained over 12 months. ClinicalTrials.gov no: NCT02505945.

PFAS and Dissolved Organic Carbon Enrichment in Surface Water Foams on a Northern U.S. Freshwater Lake.

Information is needed on the concentration of per- and polyfluoroalkyl substances (PFAS) in foams on surface waters impacted by aqueous film-forming foam (AFFF). Nine pairs of foam and underlying bulk water were collected from a single freshwater lake impacted by PFAS and analyzed for PFAS by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF) and for dissolved organic carbon (DOC). The DOC of two foam:bulk water pairs was characterized by 1H NMR. Foams were comprised of 16 PFAS with concentrations as high as 97 000 ng/L (PFOS) along with longer-chain, more hydrophobic PFAS. Only five PFAS (PFOS and shorter chain lengths) were quantified in underlying bulk waters. Enrichment factors (foam:bulk water) ranged from 10 (PFHxA) up to 2830 (PFOS). Foams impacted by AFFF gave the greatest concentrations and number of PFAS classes with PFOS concentrations exceeding the EPA health advisory level (70 ng/L). PFAS concentrations were significantly below published critical micelle concentrations and constituted <0.1% of overall DOC concentrations in foam, indicating that PFAS are a minor fraction of DOC and that DOC likely plays a central role in foam formation. Estimates indicate that foam ingestion is a potentially important route of exposure for children and adults when they are in surface waters where foam is present.

Entrance pupil masks for enhancing moiré-based distance/motion sensing.

Detection of higher-order diffraction patterns when range-finding using moiré images of fork gratings, as previously developed, is key for increased sensitivity. However, higher-order frequencies are inherently lower contrast than the fundamental frequency. Here we explore means of optically increasing contrast of moiré patterns via pupil function design. Two candidate pupil forms that increase performance above diffraction limited capabilities at specific spatial frequencies are considered, and one is chosen to proceed to experimentation. Contrast of the moiré image when using the specially designed pupil is compared to a clear aperture. Both simulation and experimental data are analyzed to show the increased contrast and sensing capabilities of the candidate entrance pupil mask.

Towards a stabilized Kerr optical frequency comb with spatial interference.

We measured a soliton's carrier frequency created by a silicon nitride Kerr comb from an interference pattern created by a spatial interferometer. The optical frequencies were determined from the interference pattern by simultaneously calibrating against an interference pattern from the pump optical frequency. Results were compared to real-time measurements by an optical spectrum analyzer (OSA). The spatial interferometer and the OSA results tracked each other, and the resulting RMS error is presented.

Altered Protein Dynamics and Increased Aggregation of Human γS-Crystallin Due to Cataract-Associated Deamidations.

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (µs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Laparoscopic magnetic sphincter augmentation versus double-dose proton pump inhibitors for management of moderate-to-severe regurgitation in GERD: a randomized controlled trial.

BACKGROUND AND AIMS: GERD patients frequently complain of regurgitation of gastric contents. Medical therapy with proton-pump inhibitors (PPIs) is frequently ineffective in alleviating regurgitation symptoms, because PPIs do nothing to restore a weak lower esophageal sphincter. Our aim was to compare effectiveness of increased PPI dosing with laparoscopic magnetic sphincter augmentation (MSA) in patients with moderate-to-severe regurgitation despite once-daily PPI therapy. METHODS: One hundred fifty-two patients with GERD, aged ≥21 years with moderate-to-severe regurgitation despite 8 weeks of once-daily PPI therapy, were prospectively enrolled at 21 U.S. sites. Participants were randomized 2:1 to treatment with twice-daily (BID) PPIs (N = 102) or to laparoscopic MSA (N = 50). Standardized foregut symptom questionnaires and ambulatory esophageal reflux monitoring were performed at baseline and at 6 months. Relief of regurgitation, improvement in foregut questionnaire scores, decrease in esophageal acid exposure and reflux events, discontinuation of PPIs, and adverse events were the measures of efficacy. RESULTS: Per protocol, 89% (42/47) of treated patients with MSA reported relief of regurgitation compared with 10% (10/101) of the BID PPI group (P < .001) at the 6-month primary endpoint. By intention-to-treat analysis, 84% (42/50) of patients in the MSA group and 10% (10/102) in the BID PPI group met this primary endpoint (P < .001). Eighty-one percent (38/47) of patients with MSA versus 8% (7/87) of patients with BID PPI had ≥50% improvement in GERD-health-related quality of life scores (P < .001), and 91% (43/47) remained off of PPI therapy. A normal number of reflux episodes and acid exposures was observed in 91% (40/44) and 89% (39/44) of MSA patients, respectively, compared with 58% (46/79) (P < .001) and 75% (59/79) (P = .065) of BID PPI patients at 6 months. No significant safety issues were observed. In MSA patients, 28% reported transient dysphagia; 4% reported ongoing dysphagia. CONCLUSION: Patients with GERD with moderate-to-severe regurgitation, especially despite once-daily PPI treatment, should be considered for minimally invasive treatment with MSA rather than increased PPI therapy. (Clinical trial registration number: NCT02505945.).

Mineral Surfaces as Agents of Environmental Proteolysis: Mechanisms and Controls.

We investigated the extent to which contact with mineral surfaces affected the molecular integrity of a model protein, with an emphasis on identifying the mechanisms (hydrolysis, oxidation) and conditions leading to protein alteration. To this end, we studied the ability of four mineral surface archetypes (negatively charged, positively charged, neutral, redox-active) to abiotically fragment a well-characterized protein (GB1) as a function of pH and contact time. GB1 was exposed to the soil minerals montmorillonite, goethite, kaolinite, and birnessite at pH 5 and pH 7 for 1, 8, 24, and 168 h and the supernatant was screened for peptide fragments using Tandem Mass Spectrometry. To distinguish between products of oxidative and hydrolytic cleavage, we combined results from the SEQUEST algorithm, which identifies protein fragments that were cleaved hydrolytically, with the output of a deconvolution algorithm (DECON-Routine) designed to identify oxidation fragments. All four minerals were able to induce protein cleavage. Manganese oxide was effective at both hydrolytic and oxidative cleavage. The fact that phyllosilicates-which are not redox active-induced oxidative cleavage indicates that surfaces acted as catalysts and not as reactants. Our results extend previous observations of proteolytic capabilities in soil minerals to the groups of phyllosilicates and Fe-oxides. We identified structural regions of the protein with particularly high susceptibility to cleavage (loops and ß strands) as well as regions that were entirely unaffected (α helix).

Passive range finder based on moiré images of Ronchi-like gratings with bilateral asymmetry.

A passive range-finding technique utilizing moiré images of Ronchi-like gratings with bilateral asymmetry, specifically fork gratings, is explored. Fork gratings are used as opposed to Ronchi gratings due to their asymmetry in the spatial and frequency domains, which enable the resolution of certain ambiguities in analysis that would otherwise be present. The two patterns which are convolved to produce the moiré image are the fork grating and the nailbed pattern of the pixelated detector. A fork grating of known size and spatial frequency is imaged with a detector of a fixed pixel pitch. Multiple analysis techniques are applied in frequency space, which lead to both relative and absolute measures of distance.

Structural and metabolic responses of Staphylococcus aureus biofilms to hyperosmotic and antibiotic stress.

Biofilms alter their metabolism in response to environmental stress. This study explores the effect of a hyperosmotic agent-antibiotic treatment on the metabolism of Staphylococcus aureus biofilms through the use of nuclear magnetic resonance (NMR) techniques. To determine the metabolic activity of S. aureus, we quantified the concentrations of metabolites in spent medium using high-resolution NMR spectroscopy. Biofilm porosity, thickness, biovolume, and relative diffusion coefficient depth profiles were obtained using NMR microimaging. Dissolved oxygen concentration was measured to determine the availability of oxygen within the biofilm. Under vancomycin-only treatment, the biofilm communities switched to fermentation under anaerobic condition, as evidenced by high concentrations of formate (7.4 ± 2.7 mM), acetate (13.1 ± 0.9 mM), and lactate (3.0 ± 0.8 mM), and there was no detectable dissolved oxygen in the biofilm. In addition, we observed the highest consumption of pyruvate (0.19 mM remaining from an initial 40 mM concentration), the sole carbon source, under the vancomycin-only treatment. On the other hand, relative effective diffusion coefficients increased from 0.73 ± 0.08 to 0.88 ± 0.08 under vancomycin-only treatment but decreased from 0.71 ± 0.04 to 0.60 ± 0.07 under maltodextrin-only and from 0.73 ± 0.06 to 0.56 ± 0.08 under combined treatments. There was an increase in biovolume, from 2.5 ± 1 mm3 to 7 ± 1 mm3 , under the vancomycin-only treatment, while the maltodextrin-only and combined treatments showed no significant change in biovolume over time. This indicated that physical biofilm growth was halted during maltodextrin-only and combined treatments.

Degradation of Glyphosate by Mn-Oxide May Bypass Sarcosine and Form Glycine Directly after C-N Bond Cleavage.

Glyphosate is the active ingredient of the common herbicide Roundup. The increasing presence of glyphosate and its byproducts has raised concerns about its potential impact on the environment and human health. In this research, we investigated abiotic pathways of glyphosate degradation as catalyzed by birnessite under aerobic and neutral pH conditions to determine whether certain pathways have the potential to generate less harmful intermediate products. Nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography (HPLC) were utilized to identify and quantify reaction products, and density functional theory (DFT) calculations were used to investigate the bond critical point (BCP) properties of the C-N bond in glyphosate and Mn(IV)-complexed glyphosate. We found that sarcosine, the commonly recognized precursor to glycine, was not present at detectable levels in any of our experiments despite the fact that its half-life (∼13.6 h) was greater than our sampling intervals. Abiotic degradation of glyphosate largely followed the glycine pathway rather than the AMPA (aminomethylphosphonic acid) pathway. Preferential cleavage of the phosphonate adjacent C-N bond to form glycine directly was also supported by our BCP analysis, which revealed that this C-N bond was disproportionately affected by the interaction of glyphosate with Mn(IV). Overall, these results provide useful insights into the potential pathways through which glyphosate may degrade via relatively benign intermediates.

Routine use of mesh during hiatal closure is safe with no increase in adverse sequelae.

BACKGROUND: Primary laparoscopic hiatal repair with fundoplication is associated with a high recurrence rate. We wanted to evaluate the potential risks posed by routine use of onlay-mesh during hiatal closure, when compared to primary repair. METHODS: Utilizing single-institutional database, we identified patients who underwent primary laparoscopic hiatal repair from January 2005 through December 2014. Retrospective chart review was performed to determine perioperative morbidity and mortality. Long-term results were assessed by sending out a questionnaire. Results were tabulated and patients were divided into 2 groups: fundoplication with hiatal closure + absorbable or non-absorbable mesh and fundoplication with hiatal closure alone. RESULTS: A total of 505 patients underwent primary laparoscopic fundoplication. Mesh reinforcement was used in 270 patients (53.5%). There was no significant difference in the 30-day perioperative outcomes between the 2 groups. No clinically apparent erosions were noted and no mesh required removal. Standard questionnaire was sent to 475 patients; 174 (36.6%) patients responded with a median follow-up of 4.29 years. Once again, no difference was noted between the 2 groups in terms of dysphagia, heartburn, long-term antacid use, or patient satisfaction. Of these, 15 patients (16.9%, 15/89) in the 'Mesh' cohort had symptomatic recurrence as compared to 19 patients (22.4%, 19/85) in the 'No Mesh' cohort (p = 0.362). A reoperation was necessary in 6 patients (6.7%) in the 'Mesh' cohort as compared to 3 patients (3.5%) in the 'No Mesh' cohort (p = 0.543). CONCLUSIONS: Onlay-mesh use in laparoscopic hiatal repair with fundoplication is safe and has similar short and long-term results as primary repair.