TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells.

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Site-Selective Unnatural Amino Acid Incorporation at Single or Multiple Positions to Control Sugar-Protein Connectivity in Glycoconjugate Vaccine Candidates.

In cellulo site-specific unnatural amino acid incorporation based on amber stop codon reassignment is a powerful tool to modify proteins at defined positions. This technique is herein applied to the selective functionalization of the Pneumococcal surface adhesin A protein at three distinct positions. Nϵ -propargyloxycarbonyl-l-lysine residues were incorporated and their alkyne groups reacted using click-chemistry with a synthetic azido-functionalized tetrasaccharide representative of one repeat unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Anti-PsaA antibody response induced in mice by the trivalent glycoconjugate was determined in comparison with corresponding monovalent and randomly functionalized conjugates. Our results suggest that controlled was superior to random conjugation for preserving antigenicity. In definitive, the reported strategy offers a unique opportunity to study the impact of carbohydrate antigen-carrier protein connectivity on immunogenicity.

Specificity of a ß-porphyranase produced by the carrageenophyte red alga Chondrus crispus and implications of this unexpected activity on red algal biology.

The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial ß-carrageenase and ß-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected ß-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0-9.0). CcGH16-3 has a KM of 4.0 ± 0.8 µM, a kcat of 79.9 ± 6.9 s-1, and a kcat/KM of 20.1 ± 1.7 µM-1 s-1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its -1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This ß-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization.

New insight into the mode of action of a GH74 xyloglucanase on tamarind seed xyloglucan: Action pattern and cleavage site.

Structural elucidation of plant cell wall xyloglucan through the analysis of enzymatically produced fragments requires detailed knowledge of enzymes hydrolytic mechanism. In this note, the mode of action and cleavage site of commercial recombinant xyloglucanases (GH74, Paenibacillus sp.) was studied on native and fluorescent-tagged tamarind xyloglucan. In complement to information provided by the manufacturer, GH74 hydrolysis was shown dual endo/exo- and exo-processive with low affinity towards labelled reducing-ends. GH74 accommodated X/G in its subsite -1 and X/L in its subsite +1. Moreover, hydrolysis kinetic indicated a GH74 activity inhibition by excess products. These results will help for application of this enzyme in xyloglucans structural analysis or for processing cell walls.

Biorefinery of apple pomace: New insights into xyloglucan building blocks.

Within the apple pomace biorefinery cascade processing framework aiming at adding value to an agroindustrial waste, after pectin recovery, this study focused on hemicellulose. The structure of the major apple hemicellulose, xyloglucan (XyG), was assessed as a prerequisite to potential developments in industrial applications. DMSO-LiCl and 4 M KOH soluble hemicelluloses from pectin-extracted apple pomace were purified by anion exchange chromatography. XyG structure was assessed by coupling xyloglucanase and endo-ß-1,4-glucanase digestions to HPAEC and MALDI-TOF MS analyses. 71.9% of pomaces hemicellulose were recovered with starch. DMSO-LiCl and 4 M KOH soluble XyG exhibited Mw of 19 and 140 kDa, respectively. Besides the XXXG, XLXG, XXLG, XXFG, XLFG and XLLG structures, novel oligosaccharides with degree of polymerization of 6-10 were observed after xyloglucanase digestion. Cellobiose and cellotriose were revealed randomly distributed in XyG backbone and were more present in DMSO-LiCl soluble XyG. Residual pomace remains a potential source of other materials.

Assessment of malreduction standards for the syndesmosis in bilateral CT scans of uninjured ankles.

AIMS: Malreduction of the syndesmosis has been reported in up to 52% of patients after fixation of ankle fractures. Multiple radiological parameters are used to define malreduction; there has been limited investigation of the accuracy of these measurements in differentiating malreduction from inherent anatomical asymmetry. The purpose of this study was to identify the prevalence of positive malreduction standards within the syndesmosis of native, uninjured ankles. METHODS: Three observers reviewed 213 bilateral lower limb CT scans of uninjured ankles. Multiple measurements were recorded on the axial CT 1 cm above the plafond: anterior syndesmotic distance; posterior syndesmotic distance; central syndesmotic distance; fibular rotation; and sagittal fibular translation. Previously studied malreduction standards were evaluated on bilateral CT, including differences in: anterior, central and posterior syndesmotic distance; mean syndesmotic distance; fibular rotation; sagittal translational distance; and syndesmotic area. Unilateral CT was used to compare the anterior to posterior syndesmotic distances. RESULTS: A difference of anterior to posterior syndesmotic distance > 2 mm was observed in 89% of ankles (n = 190) on unilateral CT assessment. Using bilateral CT, we found that 35% (n = 75) of normal ankles would be considered malreduced by current malreduction parameters. In 50 patients (23%), only one parameter was anomalous, 18 patients (8%) had two positive parameters and seven patients (3%) had three. Difference in fibular rotation had the lowest false positive rate of all parameters at 6%, whereas posterior syndesmotic distance difference had the highest at 15%. CONCLUSION: In this study, 35% of native, uninjured syndesmoses (n = 75) would be classified as malreduced by current diagnostic standards on bilateral CT and 89% had an asymmetric incisura on unilateral CT (n = 190). Current radiological parameters are insufficient to differentiate mild inherent anatomical asymmetry from malreduction of the syndesmosis. Cite this article: Bone Joint J 2021;103-B(1):178-183.

Effect of Statewide Social Distancing and Stay-At-Home Directives on Orthopaedic Trauma at a Southwestern Level 1 Trauma Center During the COVID-19 Pandemic.

OBJECTIVES: To compare orthopaedic trauma volume and mechanism of injury before and during statewide social distancing and stay-at-home directives. DESIGN: Retrospective. SETTING: Level 1 trauma center. PATIENTS/PARTICIPANTS: One thousand one hundred thirteen patients sustaining orthopaedic trauma injuries between March 17 and April 30 of years 2018, 2019, and 2020. INTERVENTION: Statewide social distancing and stay-at-home directives. MAIN OUTCOME MEASUREMENTS: Number of consults, mechanism of injury frequency, and type of injury frequency. RESULTS: During the COVID-19 pandemic, orthopaedic trauma consult number decreased. Injuries due to gunshot wounds increased and those due to automobile versus pedestrian accidents decreased. Time-to-presentation increased and length of stay decreased. Operative consults remained unchanged. CONCLUSIONS: Orthopaedic trauma injuries continued to occur during the COVID-19 pandemic at an overall decreased rate, however, with a different distribution in mechanism and type of injury. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Orthogonal Plating With a 95-Degree Blade Plate for Salvage of Unsuccessful Cephalomedullary Nailing of Atypic Femur Fractures: A Technical Trick.

Atypical femur fractures secondary to bisphosphonate use are often complicated by nonunion, which often results in progressive varus collapse, femoral head and neck bone loss, and eventual implant failure. We present a technique that uses orthogonal, dual plating of the proximal femur in addition to biologic augmentation for the management of these challenging fractures.

Evolution in Management of Tibial Pilon Fractures.

PURPOSE OF REVIEW: Tibial plafond, or pilon, fractures can be some of the most difficult fractures to manage. As they are often associated with high-energy trauma, both the soft tissue involvement and the comminuted fracture pattern pose challenges to fixation. Furthermore, the complex anatomy and trauma to the cartilage at the time of injury predispose pilon fractures to poor functional outcomes and high rates of posttraumatic arthritis. This review will discuss the recent developments in the treatment of tibial pilon fractures. RECENT FINDINGS: Historically, surgical management of pilon fractures has been associated with high rates of complications, including wound complications, infections, nonunions, and even the need for amputation. In response, staged protocols were created. However, recent studies have called this into question, demonstrating low wound complications with early definitive fixation. Additional studies are evaluating adjuvants to minimize wound complications, including the use of vancomycin powder and oxygen supplementation, while another study challenges the 7-cm myth regarding the distance needed between skin incisions. Additional research has been focused on alternative methods of managing these complex, and sometimes non-reconstructable, injuries with the use of external fixation, minimally invasive internal fixation, and primary arthrodesis. Tibial pilon fractures remain difficult to treat for even the most skilled orthopedic trauma surgeons. With improvements in surgical techniques and implants, complication rates have declined and outcomes have improved; however, the overall prognosis for these injuries often remains poor.