Long-term outcomes following pediatric endoscopic titanium ossiculoplasty: A single-institution experience.

INTRODUCTION: Endoscopic ossicular chain reconstruction (OCR) in adults has demonstrated equivalent outcomes to the traditional microscopic approach. Less data exist on endoscopic OCR outcomes in children, who have unique considerations including a smaller transcanal corridor and variable pathology. The purpose of this study was to investigate surgical and audiometric outcomes in children undergoing fully endoscopic and endoscopic-assisted OCR in both the short and long-term. METHODS: Retrospective review of all children (<17 years) who underwent endoscopic OCR at one tertiary care center between 2017 and 2021. Children undergoing primary and revision endoscopic OCR with either partial (PORP) and total ossicular reconstruction prostheses (TORP) were included. Children undergoing surgery for juvenile otosclerosis or congenital stapes fixation, or any child receiving a stapes prosthesis were excluded. Primary outcome measures were post-operative change in 4 frequency (500 Hz, 1, 2, 4 KHz) air conduction pure tone average (AC PTA) and change in air-bone gap (ABG). Secondary measures included need for readmission and/or revision surgery, complication rate, and surgery duration. RESULTS: Seventeen patients met inclusion criteria. Average age was 11.3 years (range, 5-17 years); 14 were male. A variety of fixed length, titanium total and partial prostheses were used. The most common prosthesis length was 2 mm (range 2-5 mm), and there were no intra- or perioperative complications. Mean long-term follow-up was 2.6 years. Most common pathology was congenital cholesteatoma (11/17, 64%), followed by chronic otitis media with tympanic membrane perforation (5/17, 29.4%), and extruded prosthesis (1/17, 5.9%). Intraoperatively, the most common finding was incus erosion (10/17, 58.8%), followed by malleus erosion (6/17, 35.3%), stapes erosion (4/17, 23.5%), and stapes absence (4/17, 23.5%). Eight children (47%) were reconstructed with PORPs, and 9 children (52.9%) were reconstructed with TORPs. Average ABG improved from 36.8 dB preoperatively to 19.9 dB postoperatively in the short-term and remained stable at 19.5 dB in the long-term. Average short-term ABG improvement was 4.2 dB for PORPs and 18 dB for TORPs. In the long-term, average ABG improved by 2.3 dB in PORPs and 13.4 dB in TORPs. PORPs had higher rates of ABG closure and lower AC PTAs than TORPs in the long-term. DISCUSSION: Endoscopic ossiculoplasty is a viable option in children presenting with ossicular erosion from various causes. Audiometric improvement following endoscopic partial and total ossicular reconstruction remains stable over time, with a preference towards partial in the long-term, and mirrors published outcomes for microscopic surgery.

The Role of Lumbar Drains in the Perioperative Management of Primary Spontaneous Temporal Lobe Encephaloceles and Cerebrospinal Fluid Leaks.

OBJECTIVE: To examine the role of lumbar drains (LDs) in the success of spontaneous temporal cerebrospinal fluid (CSF) leak and encephalocele repair. STUDY DESIGN: Retrospective cohort study. SETTING: Tertiary academic health system. PATIENTS: Patients undergoing repair of spontaneous temporal lobe encephaloceles or middle fossa CSF leaks during years 2017 to 2023. INTERVENTIONS: Transmastoid, middle fossa craniotomy, or combination approaches to CSF leak repair. OUTCOME MEASURES: Failure rate, complication rate, length of stay (LOS), readmission. RESULTS: Sixty-nine patients were included, with a combination approach performed in 78.3%, transmastoid in 17.4%, and isolated middle fossa craniotomy in 4.3%. Mean body mass index was 33.2, mean bony defect size width was 6.51 mm, and defect locations included the epitympanum, antrum, mastoid, and petrous apex. Multilayer closure with three or more layers was performed in 87.0%. LD was used in 73.9% of cases for a mean duration 2.27 days and was associated with longer LOS (3.27 vs. 1.56 d, p = 0.006) but not with failure rate, complications, discharge destination, or readmission. Only one major complication occurred as a result of the drain, but low-pressure headache was anecdotally common. CONCLUSIONS: Use of LD in the repair of spontaneous CSF leaks and temporal lobe encephaloceles is associated with longer LOS but not failure rates or other admission-level outcomes.

Use of Soft Cervical Collar Improves Surgeon Ergonomics During Simulated Otologic Surgery.

OBJECTIVE: To determine whether surgeon use of a soft cervical collar during endoscopic and microscopic otologic surgery is feasible and impacts surgeon ergonomics as measured by inertial sensors. STUDY DESIGN: Prospective crossover trial. SETTING: US-based otolaryngology training program. PATIENTS: Otolaryngology residents and fellows. INTERVENTIONS: Therapeutic-use of a soft cervical collar during simulated otologic surgery. MAIN OUTCOME MEASURES: Time spent in high-risk angles of neck and back flexion and extension; average angle of neck flexion, extension, rotation, and lateral bending; validated assessment of neck pain; average daily phone use. RESULTS: Fifteen subjects met criteria for inclusion. Ten of 15 (67%) were male. Seven of 15 (47%) were postgraduate year 1-2. Seven of 15 (47%) reported a history of neck pain. None reported prior spinal steroid injections or surgery. Across all subjects, use of the soft cervical collar significantly reduced time spent in high-risk angles of neck flexion/extension during both endoscopic (56% vs. 35%, p < 0.05) and microscopic (60% vs. 32%, p < 0.05) otologic surgery. There was no effect on back flexion or extension. There was no difference in time spent in high-risk neck or back angles between endoscopic and microscopic surgery. Average angles of neck or back flexion, extension, lateral bending, and rotation were not significantly different for subgroups with more operative experience, increased phone use, perception of good posture, or history of neck pain. CONCLUSIONS: Use of a soft cervical collar during simulated otologic surgery significantly reduced time spent in high-risk neck positions. These data support feasibility of soft collar use during otologic surgery and hold promise for reduction in the high rates of neck pain reported by neurotologists. PROFESSIONAL PRACTICE GAP AND EDUCATIONAL NEED: Improving surgeon ergonomics for otologic surgery. LEARNING OBJECTIVE: To identify a therapeutic intervention to mitigate neck pain in surgeons caused by assumption of high-risk cervical neck flexion and extension. DESIRED RESULT: To demonstrate that use of a readily available soft cervical collar reduces risk of neck pain in otologic surgeons. LEVEL OF EVIDENCE: II. INDICATE IRB OR IACUC: Exempt.

Step-by-step full factorial design to optimize a quantitative sandwich ELISA.

In this work, a quantitative sandwich ELISA was optimized, through a full factorial design of experiments (DOE) in successive steps of a preliminary protocol obtained by the method of one factor at a time (OFAT). The specificity of the optimized ELISA, the lower limit of quantification, the quantification range and the analytical sensitivity of the antigen quantification curve were evaluated, in comparison with the curve obtained from the preliminary protocol. The full factorial DOE was linked to a simple statistical processing, which facilitates the interpretation of the results in those laboratories where there is no trained statistician. The step-by-step optimization of the ELISA and the successive incorporation into the protocol of the best combination of factors and levels, allowed obtaining a specific immunoassay, with an analytical sensitivity 20 times greater and with a lower limit of antigen quantification that decreased from 156.25 at 9.766 ng/mL. As far as we know, there are no reports of optimization of an ELISA following the step-by-step scheme used in this work. The optimized ELISA will be used for the quantification of the TT-P0 protein, the active principle of a vaccine candidate against sea lice.

Does "Just in Time" teaching of ergonomic principles improve posture of trainees during otologic microscopic surgery?

PURPOSE: Musculoskeletal disorders are prevalent among otolaryngologists and otologists with symptoms starting during residency. Prior data suggested that high-risk joint angles were often adopted at procedure onset, suggesting a detrimental "natural" operating position. Despite its importance, dedicated ergonomic teaching is not systematically introduced into residency training. The objective of this study was to compare initial ergonomic positioning during microscopic temporal bone surgery between those who receive "Just in Time" ergonomic teaching prior to starting dissection with those who did not. MATERIALS AND METHODS: This was a pilot, prospective trial in which otolaryngology residents at an urban, multicenter tertiary care academic institution (n = 14) wore ergonomic sensors (inertial measurement units) during microscopic temporal bone lab drilling. Prior to recording, participants were randomized to receive an instructional presentation on ergonomic principles (n = 8, intervention group) or not (n = 6, control group). The inertial measurement units analyzed neck and back angles for the initial 5 min of drilling. RESULTS: Of 14 trainees, 78.6 % had prior experience with otologic microscopic cases and 14.3 % reported prior surgical ergonomic training or instruction. The groups were matched in trainee height (P = 0.54), handedness (P = 0.83), stage of otolaryngology training (P = 0.64), prior otologic microscopic surgery experience (P = 0.35), prior temporal bone drilling experience (P = 0.35), and prior teaching in ergonomic principles (P = 0.47). Junior trainees (PGY 1-3) who did not receive "Just in Time" teaching adopted a posture with significantly higher risk back flexion compared to junior trainees who received the training (25.3° vs. 5.7°, P = 0.04). There was no difference in back positioning among senior trainees (12.6° vs. -5.7°, P = 0.13). While there was a trend towards those in the intervention group adopting safer procedural posture, there was no significant difference in the overall cohort between the intervention and control groups in both neck positioning (-11.0° vs. -19.1°, P = 0.17) and back positioning (8.6° vs. 19.1°, P = 0.18). CONCLUSIONS: Musculoskeletal related pain is prevalent among otolaryngologists and otologists with data suggesting that symptoms begin during residency. Targeted "Just in Time" teaching of ergonomic principles is feasible and may be effective for development of healthy postural habits, especially among junior trainees.

Initial Experience With Two Active Transcutaneous Bone-Anchored Hearing Implants.

OBJECTIVE: To analyze our institutional experience with two active transcutaneous bone-anchored hearing aids. STUDY DESIGN: Retrospective cohort study. SETTING: Tertiary academic otology-neurotology practice. PATIENTS: Patients with conductive or mixed hearing losses meeting criteria to receive active transcutaneous bone-anchored hearing aids. INTERVENTIONS: Implantation with one of two active transcutaneous bone-anchored hearing aids. OUTCOME MEASURES: Operative time, dural exposure and decompression, use of lifts, implant position, ease of use, qualitative patient satisfaction, complication rates. RESULTS: Ten patients received Implant 1 and 11 patients received Implant 2. The most common underlying etiologies of hearing loss were chronic suppurative otitis media in 33.3%, atresia/microtia in 23.8%, and cholesteatoma in 23.8%. Average operative times were 99.3 minutes for Implant 1 and 80.9 minutes for Implant 2 ( p = 0.263). Implant 1 required lifts in 60%, dural exposure in 50%, and dural compression in 30%. Overall, placement was difficult in 47.6% of cases. There were no intraoperative complications. There were higher rates of issues with sound quality (27.3% versus 0.0%, p = 0.123) and poor cosmesis (36.4% versus 10.0%, p = 0.360) with Implant 2. Functional gain was not recorded, but all patients derived qualitative benefit from their implant. Three patients had local wound complications that self-resolved or were treated with antibiotics. One patient implanted at an outside institution required explant because of multiple infections. CONCLUSIONS: There were no statistically significant differences in outcomes comparing Implants 1 and 2; however, Implant 2 had much higher rates of issues with audio quality and poor cosmesis. Placing Implant 1 often required special techniques.

Sharing in-office otoendoscopy recordings may improve patient satisfaction: A prospective cohort study.

OBJECTIVE: Evaluate the impact of sharing otoendoscopy exams on patient satisfaction in the outpatient clinical setting. METHODS: Randomized, prospective cohort study. Consecutive adults presenting to otology clinic at one tertiary referral center were randomized into two groups: standard microscopy (SM) and video otoendoscopy (VO). The SM group had ears examined using a standard, otomicroscope; the VO group had ears examined using a 0° rigid endoscope connected to a video tower. All subjects were counseled on their exam findings in a routine manner; the VO group was concurrently shown a recording of their ear exam. All subjects completed the 18-item Patient Satisfaction Questionnaire (PSQ-18) at the conclusion of their clinic visit. RESULTS: The SM group consisted of 27 patients and VO group consisted of 23 patients. VO subjects reported higher PSQ-18 scores compared to SM subjects within the domains of communication (p = 0.04) and technical quality (p = 0.005). On linear regression models, demographic factors and positive exam findings were not predictive of patient satisfaction. CONCLUSIONS: Sharing otoendoscopy recordings may be a valuable tool that can improve patient satisfaction. PRACTICE IMPLICATIONS: Clinicians should consider sharing recordings of otoscopic exams with patients, particularly when faced with the possibility of surgery.

A Review of Noninfectious Diseases Masquerading as Acute Mastoiditis.

OBJECTIVE: Acute mastoiditis is commonly attributed to infection. Rarely do clinicians encounter cases that do not respond to traditional antibiotics or surgical management. The goal of this study was to systematically review the literature to characterize diseases masquerading as acute infectious mastoiditis. DATA SOURCES: PubMed, Embase, and Scopus. REVIEW METHODS: A systematic review was performed to identify all publications that reported on diseases with presentations mimicking acute mastoiditis, defined as postauricular redness, swelling, and tenderness. We included clinical prospective studies, retrospective studies, and case series/reports. Exclusion criteria included non-English articles, letters/commentaries, abstracts, and review articles. RESULTS: Out of 3339 results, 35 studies met final inclusion criteria. In children, 11 diseases were reported to mimic mastoiditis, including solid tumors, hematologic diseases, and autoimmune/inflammatory diseases. The most common disease in children was Langerhans cell histiocytosis, followed by rhabdomyosarcoma and acute myelogenous leukemia. In adults, 8 additional diseases were reported. The most common disease in adults was squamous cell carcinoma, followed by nasopharyngeal carcinoma and Langerhans cell histiocytosis. Presenting symptoms are reviewed, as well as characteristic radiographic, laboratory, and intraoperative features that may assist with diagnosis. A diagnostic algorithm for atypical cases of acute mastoiditis is proposed. CONCLUSION: A small but significant group of diseases in children and adults can mimic acute mastoiditis. In such cases, history and examination alone may be insufficient to reach a diagnosis, and further investigation may be necessary. Otolaryngologists should always be mindful of the possibility that noninfectious pathologies may present with a constellation of symptoms similar to mastoiditis.

Murine monoclonal antibodies against RBD of the SARS-CoV-2 spike protein as useful analytical tools for subunit vaccine development and clinical trials.

COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.

Fructooligosaccharides production by immobilized Pichia pastoris cells expressing Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase.

Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Removal of bacterial dextran in sugarcane juice by Talaromyces minioluteus dextranase expressed constitutively in Pichia pastoris.

A gene construct encoding the mature region of Talaromyces minioluteus dextranase (EC 3.2.1.11) fused to the Saccharomyces cerevisiae SUC2 signal sequence was expressed in Pichia pastoris under the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP). The increase of the transgene dosage from one to two and four copies enhanced proportionally the extracellular yield of the recombinant enzyme (r-TmDEX) without inhibiting cell growth. The volumetric productivity of the four-copy clone in fed batch fermentation (51 h) using molasses as carbon source was 1706 U/L/h. The secreted N-glycosylated r-TmDEX was optimally active at pH 4.5-5.5 and temperature 50-60 °C. The addition of sucrose (600 g/L) as a stabilizer retained intact the r-TmDEX activity after 1-h incubation at 50-60 °C and pH 5.5. Bacterial dextran in deteriorated sugarcane juice was completely removed by applying a crude preparation of secreted r-TmDEX. The high yield of r-TmDEX in methanol-free cultures and the low cost of the fed batch fermentation make the P. pastoris pGAP-based expression system appropriate for the large scale production of dextranase and its use for dextran removal at sugar mills.

2-Octyl cyanoacrylate to prevent salivary fistula formation following oral cavity microvascular reconstruction: A prospective trial.

BACKGROUND: Salivary fistulas remain a significant problem in patients undergoing major head and neck reconstructive surgery. Surgical sealants have become increasingly used in cutaneous and non-cutaneous wound closure, providing a barrier to fluids/gases and promoting healing. The purpose of this study was to determine the efficacy of a common surgical sealant, 2-Octyl Cyanoacrylate (2-OCA, Dermabond®), in the prevention of salivary fistulas following free flap reconstruction of the oral cavity. METHODS: In this non-randomized, single arm prospective trial, patients undergoing free flap reconstruction of gravity-dependent oral cavity defects were recruited. Application of 2-OCA was performed along flap inset suture lines at the time of surgery. Prospectively collected trial data were propensity score matched to a control cohort to compare outcomes. Data collected include demographics, medical co-morbidities, previous treatments, primary tumor site, and subsites reconstructed. The primary outcome measure was rate of salivary fistula formation. Secondary outcomes were time to development of leak and percentage of patients tolerating oral feeding at one month post-operatively. RESULTS: In the 46 propensity score matched pairs, eight (17.4%) out of 46 patients in the 2-OCA prospective cohort and seven (15.2%) out of 46 patients in the control cohort developed postoperative salivary fistulas within the one-month study interval (p = 1.00). The average time to postoperative leak in the 2-OCA group was 12.5 days versus 7.1 days in the control cohort (p = 0.10). In the 2-OCA group, 30 (65.2%) patients were tolerating regular diet at one month post-operatively compared to 33 (71.7%) in the control cohort (p = 0.65). CONCLUSION: Salivary fistula rates after application of a 2-OCA surgical sealant were not improved compared to a control cohort in this single institutional trial. There are several surgical sealants available, each with varying elasticity and adhesiveness. Future studies are needed to identify surgical sealants that are able to provide sufficient strength and adhesion to seal closures and combat corrosive saliva, but elastic enough to handle motion related tension during swallowing and post-operative movements in the head and neck.

Temporal Bone Encephaloceles: Utility of Preoperative Imaging.

OBJECTIVE: To determine the diagnostic efficacy and clinical value of preoperative computed tomography (CT) and magnetic resonance imaging (MRI) among patients with suspected temporal bone encephaloceles (TBE). STUDY DESIGN: Retrospective chart review from 2006 to 2018. SETTING: Tertiary referral center. SUBJECTS AND METHODS: The subjects underwent surgery for a clinically suspected TBE or cerebrospinal fluid (CSF) leak. Preoperative imaging test characteristics of CT and MRI, including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), were calculated for TBE scans and compared with intraoperative findings. RESULTS: Fifty-seven subjects with otorrhea, middle ear effusion, hearing loss, and/or meningitis with suspected TBE or CSF leak were identified. All had preoperative CT scans, and 61% (35/57) had preoperative MRI scans. Intraoperatively, 37 of 57 patients (65%) were found to have a TBE. CT scans (17% sensitivity, 100% specificity, 100% PPV, 46% NPV) were less sensitive than MRI (58% sensitivity, 100% specificity, 100% PPV, 24% NPV) for detecting TBEs. Furthermore, the time from initial scan to operative repair was significantly longer in those who had a CT followed by MRI scan compared with CT alone or a fused CT-MRI scan (mean = 68 vs 15 days, respectively; P = .004). CONCLUSION: CT and MRI provide complementary information that may aid surgical planning. However, imaging cannot always rule out TBE. In cases with high clinical suspicion, surgical confirmation is often required for definitive diagnosis and treatment. The cost of an additional preoperative study should be considered before its use.

Engineered thermostable ß-fructosidase from Thermotoga maritima with enhanced fructooligosaccharides synthesis.

The thermostable ß-fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short-chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60-70 °C). In this research, FOS produced by BfrA were characterized by HPAE-PAD analysis as a mixture of 1-kestotriose, 6G-kestotriose (neokestose), and to a major extent 6-kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non-saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized 6-kestotriose as the major component of a FOS mixture that includes minor amounts of tetra- and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3-fold higher than that of the wild-type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg-1, represents an attractive candidate for the continuous production of FOS-containing invert syrup at pasteurization temperatures.

Fluorescent Detection of Merlin-deficient Schwann Cells and Primary Human Vestibular Schwannoma Cells Using Sodium Fluorescein.

HYPOTHESIS: Merlin-deficient Schwann cells (MD-SC) and primary human vestibular schwannoma (VS) cells exhibit selective uptake of sodium-fluorescein (SF), allowing for fluorescent detection and improved visualization of tumor cells, when compared with Schwann cells (SC). BACKGROUND: SF is a fluorescent compound used for fluorescence-guided resection of gliomas. The utility of SF for VS surgery has not been assessed. METHODS: Mouse MD-SCs and rat SCs were cultured on 96-well plates at different cell densities and treated with SF at several drug concentrations and durations. Relative fluorescence units (RFU) were measured using a fluorometer to determine optimal treatment parameters in vitro. Subsequently, a four-point Likert scale for fluorescence visualization of pelleted cells was created and validated. Blinded observers rated SF-treated primary human VS and SC cultures, which were developed from deidentified specimens obtained from live and cadaveric donors, respectively. RESULTS: In contrast to SCs that showed low levels of fluorescence, MD-SCs demonstrated dose-dependent increases in RFUs when treated with incremental dosages of SF as well as longer treatment and fluorescent excitation times. In addition, RFUs were higher at greater MD-SC densities. The Likert scale for fluorescence visualization was validated using nine blinded observers and there were excellent inter- and intrarater reliabilities (intraclass coefficients of 0.989 and >0.858, respectively). Using the Likert scale, human VS treated with SF received higher scores than human SCs (p < 0.001). CONCLUSION: Mouse MD-SC and human VS cells demonstrate preferential uptake of SF when compared with normal primary SCs. Observers detected differences in fluorescence using the validated Likert scale. Further investigations into the utility of SF-guidance in VS surgery are warranted.

Fructooligosaccharides production by Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase constitutively expressed to high levels in Pichia pastoris.

The non-saccharolytic yeast Pichia pastoris was engineered to express constitutively the mature region of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Tall fescue (Schedonorus arundinaceus). The increase of the transgene dosage from one to nine copies enhanced 7.9-fold the recombinant enzyme (Sa1-SSTrec) yield without causing cell toxicity. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (38%) and extracellular release (62%) of Sa1-SSTrec to an overall activity of 102.1 U/ml when biomass reached (106 g/l, dry weight) in fed-batch fermentation using cane sugar for cell growth. The volumetric productivity of the nine-copy clone PGFT6x-308 at the end of fermentation (72 h) was 1422.2 U/l/h. Sa1-SSTrec purified from the culture supernatant was a monomeric glycoprotein optimally active at pH 5.0-6.0 and 45-50 °C. The removal of N-linked oligosaccharides by Endo Hf treatment decreased the enzyme stability but had no effect on the substrate and product specificities. Sa1-SSTrec converted sucrose (600 g/l) into 1-kestose (GF2) and nystose (GF3) in a ratio 9:1 with their sum representing 55-60% (w/w) of the total carbohydrates in the reaction mixture. Variations in the sucrose (100-800 g/l) or enzyme (1.5-15 units per gram of substrate) concentrations kept unaltered the product profile. Sa1-SSTrec is an attractive candidate enzyme for the industrial production of short-chain fructooligosaccharides, most particularly 1-kestose.

Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor

Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.

Complete sucrose hydrolysis by heat-killed recombinant Pichia pastoris cells entrapped in calcium alginate.

BACKGROUND: An ideal immobilized biocatalyst for the industrial-scale production of invert sugar should stably operate at elevated temperatures (60-70°C) and high sucrose concentrations (above 60%, w/v). Commercial invertase from the yeast Saccharomyces cerevisiae is thermolabile and suffers from substrate inhibition. Thermotoga maritima ß-fructosidase (BfrA) is the most thermoactive and thermostable sucrose-hydrolysing enzyme so far identified and allows complete inversion of the substrate in highly concentrated solutions. RESULTS: In this study, heat-killed Pichia pastoris cells bearing N-glycosylated BfrA in the periplasmic space were entrapped in calcium alginate beads. The immobilized recombinant yeast showed maximal sucrose hydrolysis at pH 5-7 and 90°C. BfrA was 65% active at 60°C and had no activity loss after incubation without the substrate at this temperature for 15 h. Complete inversion of cane sugar (2.04 M) at 60°C was achieved in batchwise and continuous operation with respective productivities of 4.37 and 0.88 gram of substrate hydrolysed per gram of dry beads per hour. The half-life values of the biocatalyst were 14 and 20 days when operated at 60°C in the stirred tank and the fixed-bed column, respectively. The reaction with non-viable cells prevented the occurrence of sucrose fermentation and the formation of by-products. Six-month storage of the biocatalyst in 1.46 M sucrose (pH 5.5) at 4°C caused no reduction of the invertase activity. CONCLUSIONS: The features of the novel thermostable biocatalyst developed in this study are more attractive than those of immobilized S. cerevisiae cells for application in the enzymatic manufacture of inverted sugar syrup in batch and fixed-bed reactors.

Analysis of 855 upper extremity fistulas created using a standard protocol: the role of graft extension to achieve functional status.

The Fistula First Breakthrough Initiative (FFBI) has been one of the most important national programs to help achieve considerable improvements in the care of patients on chronic hemodialysis. FFBI has helped place guidelines to push practitioners to reduce the use of tunneled central venous catheters and to increase the rate of arteriovenous fistula use in patients requiring chronic hemodialysis access. However, despite current guidelines, no specific protocols exist for the creation and management of autogenous arteriovenous fistulas and outcomes at most centers are below national benchmarks. In this study, we examine the effectiveness of a standard protocol used at our institution for the creation of autogenous upper extremity fistulas for hemodialysis access in achieving early cannulation and early removal of tunneled dialysis catheters. Our review encompasses 855 consecutive autogenous fistulas created over a 10-year period. Our findings suggest that the use of a standard protocol for creation and management of autogenous fistulas can help increase the rate of functional accesses over national benchmarks. Additionally, extension/conversion of malfunctioning fistulas to grafts appears to be an excellent method to expedite removal of a tunneled dialysis catheter with concomitant preservation of a fistula.

Constitutive high-level expression of a codon-optimized ß-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris.

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable ß-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.