Association between nociplastic pain and premature endocrine therapy discontinuation in breast cancer patients.

PURPOSE: At least 5 years of adjuvant endocrine therapy (ET) is recommended for patients with hormone receptor-positive invasive breast cancer to reduce cancer recurrence risk. Up to half of patients prematurely discontinue ET, often due to musculoskeletal pain. Nociplastic pain is abnormal central nervous system pain processing without evidence of tissue or neuronal damage. This study aimed to evaluate the relationship between baseline nociplastic pain and ET discontinuation. METHODS: This was a retrospective, single center, cohort study. Included patients were female, had stage 0-III invasive breast cancer, did not receive neoadjuvant therapy, and completed quality of life questionnaires prior to breast surgery, including Fibromyalgia Survey for nociplastic pain. Clinical data including duration of ET were abstracted from the medical record. Patient characteristics were analyzed with t-tests and Chi-squared tests, as appropriate. Univariate and multivariable regressions were performed with Cox proportional hazard models. RESULTS: Six hundred eighty-one patients diagnosed between 2012 and 2019 met inclusion criteria; 480 initiated ET and were included in the analysis. Of these 480 patients, 203 (42.3%) prematurely discontinued initial ET therapy. On univariate analysis, tamoxifen use (hazard ratio [HR] 0.70, p = 0.021) and premenopausal status (HR 0.73, p = 0.04) were inversely associated with ET discontinuation, while Fibromyalgia Score was positively associated (HR 1.04, p = 0.043). On multivariable analysis, baseline Fibromyalgia Score remained associated with ET discontinuation. CONCLUSION: Nociplastic pain present prior to surgery was associated with premature ET discontinuation. Fibromyalgia Score screening may be useful for evaluating ET discontinuation risk. Treatments targeting nociplastic pain may be more effective for treating ET-emergent pain.

Dynamic enrichment of Bayesian small-sample, sequential, multiple assignment randomized trial design using natural history data: a case study from Duchenne muscular dystrophy.

In Duchenne muscular dystrophy (DMD) and other rare diseases, recruiting patients into clinical trials is challenging. Additionally, assigning patients to long-term, multi-year placebo arms raises ethical and trial retention concerns. This poses a significant challenge to the traditional sequential drug development paradigm. In this paper, we propose a small-sample, sequential, multiple assignment, randomized trial (snSMART) design that combines dose selection and confirmatory assessment into a single trial. This multi-stage design evaluates the effects of multiple doses of a promising drug and re-randomizes patients to appropriate dose levels based on their Stage 1 dose and response. Our proposed approach increases the efficiency of treatment effect estimates by (i) enriching the placebo arm with external control data, and (ii) using data from all stages. Data from external control and different stages are combined using a robust meta-analytic combined (MAC) approach to consider the various sources of heterogeneity and potential selection bias. We reanalyze data from a DMD trial using the proposed method and external control data from the Duchenne Natural History Study (DNHS). Our method's estimators show improved efficiency compared to the original trial. Also, the robust MAC-snSMART method most often provides more accurate estimators than the traditional analytic method. Overall, the proposed methodology provides a promising candidate for efficient drug development in DMD and other rare diseases.

Arthroscopic Biceps Transfer to the Glenoid With Bankart Repair Grants Satisfactory 2-Year Results for Recurrent Anteroinferior Glenohumeral Instability in Subcritical Bone Loss.

PURPOSE: To evaluate the short-term outcomes of the arthroscopic dynamic anterior stabilization (DAS), which is a transfer of the intra-articular portion of the long head biceps through the subscapularis split and fixation on the anterior glenoid, combined with a Bankart repair. METHODS: A retrospective evaluation was performed of DAS and a minimum of 2-year follow-up. Inclusion criteria were the presence of anteroinferior instability, a positive apprehension test at 90° of abduction and external rotation, and subcritical glenoid bone loss (less than 20%). Exclusion criteria were severe (≥20%) glenoid bone loss, presence of biceps lesions or rupture (spontaneous or biceps tenotomy), pre-existing glenohumeral osteoarthritis, multidirectional or voluntary instability, previous arthroscopic stabilization procedure, and epilepsy. Outcomes included the Rowe score, range of motion (ROM), and recurrence. RESULTS: Twenty-three patients were treated with DAS and arthroscopic Bankart repair during the study period. One person was lost to follow-up, leaving 22 patients available at last follow-up. Those 22 patients had an average age of 31.9 ± 12.3 years (range, 18-68) and were evaluated at an average follow-up of 3.2 ± 0.7 years (range, 1.2-4.2). The Rowe score increased from 36.1 ± 16.2 (range, 10-70) preoperatively to 89.8 ± 20.1 (range, 30-100) postoperatively (P < .001) with almost all patients (90.9%) improving their score beyond the minimal clinically important difference of 9.7 points. Postoperatively, ROM was maintained. Three patients (13.6%) analyzed at final follow-up demonstrated recurrence, one was successfully treated conservatively, but two revised with a Latarjet. No postoperative Popeye deformity, biceps cramping, or other complication were reported. CONCLUSIONS: The DAS procedure may be an option for augmentation of a Bankart repair in patients with anterior shoulder instability and subcritical bone loss. ROM is maintained without evidence of postoperative Popeye deformity or biceps cramping. LEVEL OF EVIDENCE: Level IV, retrospective study.

Insights into the Catalytic Mechanism of a Novel XynA and Structure-Based Engineering for Improving Bifunctional Activities.

Xylan and cellulose are the two major constituents of numerous types of lignocellulose. The bifunctional enzyme that exhibits xylanase/cellulase activity has attracted a great deal of attention in biofuel production. Previously, a thermostable GH10 family enzyme (XynA) from Bacillus sp. KW1 was found to degrade both xylan and cellulose. To improve bifunctional activity on the basis of structure, we first determined the crystal structure of XynA at 2.3 Å. Via molecular docking and activity assays, we revealed that Gln250 and His252 were indispensable to bifunctionality, because they could interact with two conserved catalytic residues, Glu182 and Glu280, while bringing the substrate close to the activity pocket. Then we used a structure-based engineering strategy to improve xylanase/cellulase activity. Although no mutants with increased bifunctional activity were obtained after much screening, we found the answer in the N-terminal 36-amino acid truncation of XynA. The activities of XynA_ΔN36 toward beechwood xylan, wheat arabinoxylan, filter paper, and barley ß-glucan were significantly increased by 0.47-, 0.53-, 2.46-, and 1.04-fold, respectively. Furthermore, upon application, this truncation released more reducing sugars than the wild type in the degradation of pretreated corn stover and sugar cane bagasse. These results showed the detailed molecular mechanism of the GH10 family bifunctional endoxylanase/cellulase. The basis of these catalytic performances and the screened XynA_ΔN36 provide clues for the further use of XynA in industrial applications.

Active Sites Engineering toward Superior Carbon-Based Oxygen Reduction Catalysts via Confinement Pyrolysis.

Developing efficient and low-cost defective carbon-based catalysts for the oxygen reduction reaction (ORR) is essential to metal-air batteries and fuel cells. Active sites engineering toward these catalysts is highly desirable but challenging to realize boosted catalytic performance. Herein, a sandwich-like confinement route to achieve the controllable regulation of active sites for carbon-based catalysts is reported. In particular, three distinct catalysts including metal-free N-doped carbon (NC), single Co atoms dispersed NC (Co-N-C), and Co nanoparticles-contained Co-N-C (Co/Co-N-C) are controllably realized and clearly identified by synchrotron radiation-based X-ray spectroscopy. Electrochemical measurements suggest that the Co/Co-N-C catalyst delivers optimized ORR performance due to the rich Co-Nx active sites and their synergistic effect with metallic Co nanoparticles. This work provides deep insight for rationally designing efficient ORR catalyst based on active sites engineering.

Two-dimensional peripheral refraction in adults.

Peripheral refraction has been studied for decades; however, its detection and description are somehow simplistic and limited. Therefore, their role in visual function and refractive correction, as well as myopia control, is not completely understood. This study aims to establish a database of two-dimensional (2D) peripheral refraction profiles in adults and explore the features for different central refraction values. A group of 479 adult subjects were recruited. Using an open-view Hartmann-Shack scanning wavefront sensor, their right naked eyes were measured. The overall features of the relative peripheral refraction maps showed myopic defocus, slight myopic defocus, and hyperopic defocus in the hyperopic and emmetropic groups, in the mild myopic group, and in other myopic groups, respectively. Defocus deviations with central refraction vary in different regions. The defocus asymmetry between the upper and lower retinas within 16° increased with the increase of central myopia. By characterizing the variation of peripheral defocus with central myopia, these results provide rich information for possible individual corrections and lens design.

Muscle Edema of Retraction and Pseudo-Fatty Infiltration After Traumatic Rotator Cuff Tears: An Experimental Model in Sheep.

Background: Traumatic rotator cuff tears can result in retraction of the tendon and may be associated with muscle edema, which may be confused with fatty infiltration as seen on magnetic resonance imaging (MRI). Purpose: To describe the characteristics of a type of edema associated with acute retraction of the rotator cuff tendon (termed "edema of retraction") and to highlight the risk of mistaking it with pseudo-fatty infiltration of the rotator cuff muscle. Study Design: Descriptive laboratory study. Methods: A total of 12 alpine sheep were used for analysis. On the right shoulder, osteotomy of the greater tuberosity was performed to release the infraspinatus tendon; the contralateral limb acted as the control. MRI was performed immediately after surgery (time zero) and at 2 and 4 weeks postoperatively. T1-weighted, T2-weighted, and Dixon pure-fat sequences were reviewed for hyperintense signals. Results: Edema of retraction resulted in hyperintense signals around or within the retracted rotator cuff muscle on both T1- and T2-weighted imaging, but there was an absence of hyperintense signals on Dixon pure-fat imaging. This represented pseudo-fatty infiltration. Edema of retraction created a characteristic "ground glass" appearance of the muscle on T1-weighted sequences and was often found in either the perimuscular or intramuscular location of the rotator cuff muscle. Compared to time zero values, a decrease in the percentage of fatty infiltration was observed at 4 weeks postoperatively (16.5% ± 4.0% vs 13.8% ± 2.9%, respectively; P < .005). Conclusion: The location of edema of retraction was often peri- or intramuscular. Edema of retraction presented as a characteristic "ground glass" appearance of the muscle on T1-weighted sequences and led to a decrease in the fat percentage because of a dilution effect. Clinical Relevance: Physicians should be aware that this edema can result in a form of pseudo-fatty infiltration, as it is associated with hyperintense signals on both T1- and T2-weighted sequences, and it can be mistaken for fatty infiltration.

Characterization of two novel highly active glycoside hydrolase family 53 endo-1,4-ß-galactanases and their synergism with other carbohydrases in plant polysaccharide decomposition.

Two novel Bacillus glycoside hydrolase family 53 (GH53) endo-1,4-ß-galactanases (Bs936 and Bs4828) were identified. The recombinant Bs936 and Bs4828 displayed maximal activities at pH 5.5/55 °C and pH 6.5/65 °C, respectively. Stability analyses revealed that the enzymes were stable between pH 4.5-10, retaining over 80% activities after 12 h incubation at 25 °C. Moreover, Bs936 maintained about 75% activity after being treated at 45 °C for 2 h, while Bs4828 kept full activity after 4 h of incubation at 50 °C. Importantly, Bs936 and Bs4828 exhibited good activity towards ß-1,4-galactan, showing specific activities of 1859.46- and 3110.79 U/mg towards potato galactan, respectively. The corresponding Km and kcat/Km were 4.93 mg/mL and 296.35 mL/mg/s for Bs936, and 6.60 mg/mL and 707.12 mL/mg/s for Bs4828. Extensive synergy assays revealed that each endo-1,4-ß-galactanase acted synergistically with the other ß-galactosidase (Bs937 or Bs4829), endo-1,5-α-L-arabinosidase (BsAbn2) and an endoxylanase/endoglucanase+cellulase mixture (BaXynA + LsCel) towards the hydrolysis of galactan, debranched arabinan/arabinan, and pretreated corn stover, respectively. The corresponding degrees of synergy were 1.28, 1.17/1.09, and 1.15 for Bs486 and 1.55, 1.04/1.08, and 1.11 for Bs4828, respectively. This study expands the repertoire of GH53 enzymes, and demonstrates the role of two highly active endo-1,4-ß-galactanases in polysaccharides hydrolysis.

Achilles Tendon-Bone Block Allograft for Massive Rotator Cuff Tears With Bony Deficiency of the Greater Tuberosity: A Minimum 2-Year Follow-up Study.

BACKGROUND: Massive rotator cuff tears associated with greater tuberosity bone loss are challenging to treat. Repairing the rotator cuff without addressing the greater tuberosity deficiency may result in poorer clinical outcomes. HYPOTHESIS: Utilizing an Achilles tendon-bone block allograft to address both the massive rotator cuff tear and greater tuberosity bone loss concurrently can result in improved clinical outcomes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: The authors performed a retrospective study of patients treated between January 2011 and December 2018 with Achilles tendon-bone block allograft for massive rotator cuff tears associated with greater tuberosity bone loss. The inclusion criteria were massive rotator cuff tear and bony deficiency of the greater tuberosity; patients with a history of bony metabolism disease, connective tissue disease, and previous surgery to the wrist or elbow of the affected limb were excluded. Range of motion, visual analog scale for pain, Constant score, and Single Numeric Assessment Evaluation score were assessed preoperatively and at a minimum of 2 years postoperatively. Radiographs and ultrasound images were evaluated to assess allograft union and rotator cuff integrity. RESULTS: Five patients (3 male and 2 female; mean age, 54.0 ± 12.2 years) were included in the study. The mean follow-up was 80.6 ± 33.7 months. Preoperative to postoperative values improved significantly on the visual analog scale (from 45.8 ± 25.5 to 14.5 ± 14.1; P = .04), Constant score (from 36.8 ± 7.9 to 73.5 ± 3.1; P < .001), and Single Numeric Assessment Evaluation score (from 42.5 ± 26.3 to 82.5 ± 10.4; P = .04). Forward flexion improved significantly from 53° ± 47° to 149° ± 17° (P = .03). Four of the 5 patients achieved bony union. One patient required removal of symptomatic hardware at 6 months postoperatively, and 1 patient required revision surgery at 1 year postoperatively because of progressive osteonecrosis of the humeral head. CONCLUSION: In patients with massive rotator cuff tears and a greater tuberosity bony deficiency, utilizing an Achilles tendon-bone block allograft to restore the bony defect and reinforce the rotator cuff repair was safe and effective. At a minimum of 2 years postoperatively, most patients demonstrated improved clinical outcomes, tendon healing, and graft incorporation.

Molecular and Biochemical Analyses of a Novel Trifunctional Endoxylanase/Endoglucanase/Feruloyl Esterase from the Human Colonic Bacterium Bacteroides intestinalis DSM 17393.

A novel enzyme Bi76 comprising GH10, E_set_Esterase_N, and CE1 modules was identified, with the highest homology (62.9%) with a bifunctional endoxylanase/feruloyl esterase among characterized enzymes. Interestingly, Bi76 hydrolyzed glucan substrates besides xylans and feruloylated substrates, suggesting that it is the first characterized trifunctional endoxylanase/endoglucanase/feruloyl esterase. Analyses of truncation variants revealed that GH10 and E_set_Esterase_N + CE1 modules encoded endoxylanase/endoglucanase and feruloyl esterase activities, respectively. Synergism analyses indicated that endoxylanase, α-l-arabinofuranosidase, and feruloyl esterase acted cooperatively in releasing ferulic acid (FA) and xylooligosaccharides from feruloylated arabinoxylan. The interdomain synergism of Bi76 overmatched the intermolecular synergism of TM1 and TM2. Importantly, Bi76 exhibited good capacity in producing FA, releasing 5.20, 4.38, 2.12, 1.35, 0.46, and 0.19 mg/g from corn bran, corn cob, wheat bran, corn stover, rice husk, and rice bran, respectively. This study expands the trifunctional endoxylanase/endoglucanase/feruloyl esterase repertoire and demonstrates the great potential of Bi76 in agricultural residue utilization.

Reconstruction of the Sternoclavicular Joint After Excessive Medial Clavicle Resection.

Medial clavicle excision is a rarely indicated procedure and may be performed in different pathologies affecting the medial clavicle. Excessive medial clavicle resection with an injury to the costoclavicular ligament often leads to poor postoperative results. The exact surgical treatment used in this kind of pathology when conservative treatment is unsuccessful remains unclear. The aim of this Technical Note is to describe our preferred surgical technique to treat this condition.

Capsulolabral Reconstruction During the Open Latarjet Procedure.

Surgical treatment of anterior glenohumeral joint instability can be challenging and carries the inherent risk of recurrent instability, dislocation arthropathy, and postoperative loss of external rotation. In the current manuscript, a technique for combined reconstruction of anterior labrum and capsule, with concomitant reduction of the humeral head during anterior capsule reconstruction in open Latarjet procedure, is presented. Analogous to other techniques, the coracoid graft is fixed on the anteroinferior part of the glenoid between 3 and 5 o'clock. However, for this technique, reattachment of the labrum is performed between the native glenoid and the bone graft. Additionally, during the reconstruction of the anterior capsule on the coracoacromial ligament, while the operated arm is held in external rotation to avoid the postoperative rotational deficit, the humeral head is reduced posteriorly in the center of the glenoid during adduction, slight anterior forward flexion, and a posterior lever push. By doing so, the inherent theoretical risks of persistent instability and dislocation arthropathy are believed to be decreased. Further studies are needed to clarify the long-term consequences of this surgical technique in the clinical setting.

Reverse Shoulder Arthroplasty with Bony and Metallic versus Standard Bony Reconstruction for Severe Glenoid Bone Loss. A Retrospective Comparative Cohort Study.

There are different techniques to address severe glenoid erosion during reverse shoulder arthroplasty (RSA). This study assessed the clinical and radiological outcomes of RSA with combined bony and metallic augment (BMA) glenoid reconstruction compared to bony augmentation (BA) alone. A review of patients who underwent RSA with severe glenoid bone loss requiring reconstruction from January 2017 to January 2019 was performed. Patients were divided into two groups: BMA versus BA alone. Clinical outcome measurements included two years postoperative ROM, Constant score, subjective shoulder value (SSV), and the American Shoulder and Elbow Surgeons Shoulder (ASES) score. Radiological outcomes included radiographic evidence of scapular complications and graft incorporation. The BMA group had significantly different glenoid morphology (p < 0.001) and greater bone loss thickness than the BA group (16.3 ± 3.8 mm vs. 12.0 ± 0.0 mm, p = 0.020). Both groups had significantly improved ROM (anterior forward flexion and external rotation) and clinical scores (Constant, SSV and ASES scores) at 2 years. Greater improvement was observed in the BMA group in terms of anterior forward flexion (86.3° ± 27.9° vs. 43.8° ± 25.6°, p = 0.013) and Constant score (56.6 ± 10.1 vs. 38.3 ± 16.7, p = 0.021). The BA group demonstrated greater functional and clinical improvements with higher postoperative active external rotation and ASES results (active external rotation, 49.4° ± 17.0° vs. 29.4° ± 14.7°, p = 0.017; ASES, 89.1 ± 11.3 vs. 76.8 ± 11.0, p = 0.045). The combination use of bone graft and metallic augments in severe glenoid bone loss during RSA is safe and effective and can be considered in cases of severe glenoid bone loss where bone graft alone may be insufficient.

Biochemical analyses of a novel thermostable GH5 endo ß-1,4-mannanase with minor ß-1,4-glucosidic cleavage activity from Bacillus sp. KW1 and its synergism with a commercial α-galactosidase on galactomannan hydrolysis.

A novel GH5 endo-1,4-ß-mannanase (BaMan5A) was identified from Bacillus sp. KW1, it shares the highest sequence identity (86%) with another characterized Bacillus endo-1,4-ß-mannanase. The recombinant BaMan5A displayed maximum activity at pH 7.0 and 70 °C, it was stable at a broad pH range (pH 3.5-11.0) after 12-h incubation at 25 °C, and exhibited good thermostability, retaining about 100% and 85% activity after incubating at 60 °C for 12 h and 65 °C for 8 h, respectively. The results of polysaccharide hydrolysis revealed that the enzyme can only hydrolyze mannan substrates, including carob galactomannan, konjac glucomannan, 1,4-ß-D-mannan, locust bean gum, and guar gum, yielding mannose, mannobiose, mannotriose, and some other oligosaccharides. The best substrate was carob galactomannan, the corresponding specific activity and Km value were 10,886 µmol/min/µmol and 3.31 mg/mL, respectively. Interestingly, BaMan5A was capable to hydrolyze both manno-oligosaccharides and cello-oligosaccharides, including mannotetraose, mannopentaose, mannohexaose, cellopentaose and cellohexaose. Furthermore, BaMan5A acted synergistically with a commercial α-galactosidase (CbAgal) on galactomannan depolymerization, a best synergy degree of 1.58 was achieved after optimizing enzyme ratios. This study not only expands the diversity of Bacillus GH5 ß-mannanase, but also discloses the potential of BaMan5A in industrial application.

Peptide-mediated porphyrin based hierarchical complexes for light-to-chemical conversion.

We present a new strategy for the biomimetic preparation of integrated photoactive complexes consisting of light harvesting and electron separation/transfer components via the hierarchical assembly of porphyrin and platinum nanoparticles on the surface of short-peptide self-assembled structures. The complexes can catalyze the conversion of visible light energy into chemical energy in the absence of an electron mediator and store it as nicotinamide adenine dinucleotide (NADH). This provides a promising step towards artificial photosystems through precisely controlled interactions of light-capturing agents, electron separators and biomolecules.

Two-Dimensional, High-Resolution Peripheral Refraction in Adults with Isomyopia and Anisomyopia.

Purpose: The purpose of this study was to investigate the two-dimensional peripheral refraction in fellow eyes of patients with isomyopia and anisomyopia. Methods: Sixty-eight young adults were recruited, including 25 isomyopes with interocular differences (IODs) of foveal refraction < 1.00 D and 43 anisomyopes with IOD > 1.50 D. Peripheral refraction across an area of the visual field of 60° × 36° with a resolution of 1° was measured using a custom-made Hartmann-Shack wavefront sensor. The retinal area was divided into 3 × 3 zones for comparison between the fellow eyes. Results: There was no difference of refraction in all corresponding zones between the fellow eyes in the isomyopic group (all P > 0.05). The IODs between more myopic (MM) eyes and less myopic (LM) eyes in the anisomyopic group ranged from -1.40 to approximately -2.46 D (all P <0.001), which was flagged in the center and attenuated in peripheral zones by varied magnitudes. In the stratification analysis for different levels of anisomyopia, the nasal retina first presented significant relative hyperopic shifts compared to the center, followed by the temporal retina. In contrast, the superior and inferior periphery only differed from the center when the central IOD was greater than 3.00 D. Conclusions: The two-dimensional peripheral refraction patterns showed a mirror symmetry between the fellow eyes of a patient with isomyopia. However, in the anisomyopic group, peripheral refraction showed significantly relative hyperopic shift when compared with the center and developed with a varied rate in different areas. These findings may indicate an asymmetrical variation in the peripheral refraction patterns during myopia progression.

A novel glycoside hydrolase family 42 enzyme with bifunctional ß-galactosidase and α-L-arabinopyranosidase activities and its synergistic effects with cognate glycoside hydrolases in plant polysaccharides degradation.

GH42 enzymes are potential candidates for bifunctional ß-galactosidase/α-L-arabinopyranosidase. A novel GH42 enzyme (BaBgal42A) from Bacillus was identified, the recombinant BaBgal42A hydrolyzed not only ß-D-galactopyranosidic bonds in pNP-ß-D-galactopyranoside, oNP-ß-D-galactopyranoside, lactose, galactan, and arabinan but also α-L-arabinopyranosidic linkages in pNP-α-L-arabinopyranoside, wheat arabinoxylan and galactan. The Km values of BaBgal42A for pNP-ß-D-galactopyranoside and pNP-α-L-arabinopyranoside were 2.76 and 16.23 mM, respectively. Investigation of cooperative activities of BaBgal42A with cognate enzymes revealed that BaBgal42A showed obvious synergy with an endo-ß-1,4-galactanase (BaGal53A) in the decomposition of galactan, supplementing BaBgal42A resulted in a 0.56-fold increase in the release of reducing sugars; BaBgal42A also exhibited a little synergy with its cognate endoxylanase (BaXynA)/α-L-arabinofuranosidase (BaAraA) in hydrolyzing wheat arabinoxylan/arabinan, addition of BaBgal42A released 12.7%/7.8% more reducing sugars than that produced by BaXynA/BaAraA alone. Moreover, BaBgal42A is a cold-adapted enzyme, exhibiting 28-46% of the maximal activity at the range of 5-20 °C and its activity was slightly stimulated by addition of Na+, K+, or Ca2+ at low concentrations. This study not only expands the diversity within GH42 family, but also provides new insights into the role of microbial GH42 enzymes, which would contribute to its potential application in polysaccharides degradation and milk lactose hydrolysis.

A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis.

BACKGROUND: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. RESULTS: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65 °C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25 °C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60 °C for 60 h, and its half-lives at 65 °C and 70 °C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-ß-d-cellobioside (pNPC), and p-nitrophenyl-ß-d-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. CONCLUSIONS: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.

Characterization of the cross-linked enzyme aggregates of a novel ß-galactosidase, a potential catalyst for the synthesis of galacto-oligosaccharides.

A novel ß-galactosidase (Bgal1-3) was isolated from a marine metagenomic library and then its cross-linked enzyme aggregates (CLEAs) were prepared. The enzymatic properties of Bgal1-3-CLEAs were studied and compared with that of the free enzyme. The thermostability and storage stability of Bgal1-3 were significantly improved after it was immobilized as CLEAs. The galactose-tolerance of the enzyme was also enhanced after the immobilization, which could relieve the inhibitory effect and then tends to be beneficial for the galacto-oligosaccharides (GOS) synthesis. Moreover, higher GOS yield was achieved (59.4 ± 1.5%) by Bgal1-3-CLEAs compared to the free counterpart (57.1 ± 1.7%) in an organic-aqueous biphasic system. The GOS content and composition of the syrups synthesized by the free enzyme and Bgal1-3-CLEAs were similar and they both contained at least seven different oligosaccharides with the degree of polymerization (DP) ranging between 3 and 9. Furthermore, Bgal1-3-CLEAs maintained 82.1 ± 2.1% activity after ten cycles of reuse; the GOS yield of the tenth batch was 52.3 ± 0.3%, which was still higher than that of the most former reports. To the best of our knowledge, this is the first report on the GOS synthesis using CLEAs of ß-galactosidase in an organic-aqueous biphasic system. The study not only further expands the application scope of CLEA, but also provides a potential catalyst for the synthesis of GOS with low cost.