Biochemical characterization of a novel acid-active endopolygalacturonase for pectin depolymerization, pectic-oligomer production, and fruit juice clarification.

Endopolygalacturonases are crucial pectinases known for their efficient and sustainable pectin depolymerization activities. The present study identified a novel gene encoding endopolygalacturonase from an acidic mine tailing metagenome. The putative gene showed a maximum identity of 67.55 % with an uncharacterized peptide sequence from Flavobacterium fluvii. The gene was cloned and expressed in a heterologous host, E. coli. Biochemical characterization of the novel endopolygalacturonase enzyme variant (EPHM) showed maximum activity at 60 °C and at 5.0 pH, while retaining 50 % activity under the temperature and pH range of 20 °C to 70 °C for 6 h, and 3.0 to 10.0 for 3 h, respectively. The enzyme exhibited tolerance to different metal ions. EPHM was characterized for the depolymerization of methylated pectin into pectic oligosaccharides. Further, its utility was established for fruit juice clarification, as endorsed by high transmittance, significant viscosity reduction, and release of reducing sugars in the treated fruit juice samples.

A novel thermotolerant L-rhamnose isomerase variant for biocatalytic conversion of D-allulose to D-allose.

A novel L-rhamnose isomerase was identified and cloned from an extreme-temperature aquatic habitat metagenome. The deduced amino acid sequence homology suggested the possible source of this metagenomic sequence to be Chloroflexus islandicus. The gene expression was performed in a heterologous host, Escherichia coli, and the recombinant protein L-rhamnose isomerase (L-RIM) was extracted and purified. The catalytic function of L-RIM was characterized for D-allulose to D-allose bioconversion. D-Allose is a sweet, rare sugar molecule with anti-tumour, anti-hypertensive, cryoprotective, and antioxidative properties. The characterization experiments showed L-RIM to be a Co++- or Mn++-dependent metalloenzyme. L-RIM was remarkably active (~ 80%) in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges. Optimal L-RIM activity with D-allulose as the substrate occurred at pH 7.0 and 75 °C. The enzyme was found to be excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively. L-RIM catalysis conducted at slightly acidic pH of 6.0 and 70 °C achieved biosynthesis of about 30 g L-1 from 100 g L-1 D-allulose in 3 h. KEY POINTS: • The present study explored an extreme temperature metagenome to identify a novel gene that encodes a thermostable l-rhamnose isomerase (L-RIM) • L-RIM exhibits substantial (80% or more) activity in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges • L-RIM is excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively.

D-Allulose 3-epimerase of Bacillus sp. origin manifests profuse heat-stability and noteworthy potential of D-fructose epimerization.

BACKGROUND: D-Allulose is an ultra-low calorie sugar of multifarious health benefits, including anti-diabetic and anti-obesity potential. D-Allulose 3-epimerase family enzymes catalyze biosynthesis of D-allulose via epimerization of D-fructose. RESULTS: A novel D-allulose 3-epimerase (DaeB) was cloned from a plant probiotic strain, Bacillus sp. KCTC 13219, and expressed in Bacillus subtilis cells. The purified protein exhibited substantial epimerization activity in a broad pH spectrum, 6.0-11.0. DaeB was able to catalyze D-fructose to D-allulose bioconversion at the temperature range of 35 °C to 70 °C, exhibiting at least 50 % activity. It displaced excessive heat stability, with the half-life of 25 days at 50 °C, and high turnover number (kcat 367 s- 1). The coupling of DaeB treatment and yeast fermentation of 700 g L- 1 D-fructose solution yielded approximately 200 g L- 1 D-allulose, and 214 g L- 1 ethanol. CONCLUSIONS: The novel D-allulose 3-epimerase of Bacillus sp. origin discerned a high magnitude of heat stability along with exorbitant epimerization ability. This biocatalyst has enormous potential for the large-scale production of D-allulose.

Valproate-Induced Lower Extremity Swelling.

New-onset severe peripheral edema warrants an extensive evaluation, including congestive heart failure, cirrhosis, nephrotic syndrome, hypoalbuminemia, or lymphatic or venous obstruction.

Salmonella Prostatic Abscess in an Immunocompetent Patient.

Patients returning from travel to a foreign country often present with atypical infections that can present as diagnostic challenges. Although rarely seen in the United States, Salmonella infections are commonly seen in Sub-Saharan Africa. The common clinical manifestations of Salmonella infection include fever and diarrhea; however, about 5% of cases of non-typhi Salmonella progress to bacteremia. Here, we present a case of a unique presentation of Salmonella infection manifesting as a prostatic abscess in an immunocompetent patient.

A Novel d-Allulose 3-Epimerase Gene from the Metagenome of a Thermal Aquatic Habitat and d-Allulose Production by Bacillus subtilis Whole-Cell Catalysis.

A novel d-allulose 3-epimerase gene (daeM) has been identified from the metagenomic resource of a hot-water reservoir. The enzyme epimerizes d-fructose into d-allulose, a functional sugar of rare abundance in nature. The metagenomic DNA fragment was cloned and expressed in Escherichia coli The purified recombinant protein (DaeM) was found to be metal dependent (Co2+ or Mn2+). It displayed the maximal levels of catalytic activity in a pH range of 6 to 11 and a temperature range of 75°C to 80°C. The enzyme exhibited remarkably high thermal stability at 60°C and 70°C, with half-life values of 9,900 and 3,240 min, respectively. To the best of our knowledge, this is the highest thermal stability demonstrated by a d-allulose 3-epimerase that has been characterized to date. The enzymatic treatment of 700 mg·ml-1 d-fructose yielded about 217 mg·ml-1 d-allulose, under optimal condition. The catalytic product was purified, and its nuclear magnetic resonance (NMR) spectra were found to be indistinguishable from those of standard d-allulose. For biomolecule production, the whole-cell catalysis procedure avoids the tedious process of extraction and purification of enzyme and also offers better biocatalyst stability. Further, it is desirable to employ safe-grade microorganisms for the biosynthesis of a product. The daeM gene was expressed intracellularly in Bacillus subtilis A whole-cell catalysis reaction performed with a reaction volume of 1 liter at 60°C yielded approximately 196 g·liter-1 d-allulose from 700 g·liter-1 d-fructose. Further, the whole recombinant cells were able to biosynthesize d-allulose in apple juice, mixed fruit juice, and honey.IMPORTANCE d-Allulose is a noncaloric sugar substitute with antidiabetes and antiobesity potential. With several characteristics of physiological significance, d-allulose has wide-ranging applications in the food and pharmacology industries. The development of a thermostable biocatalyst is an objective of mainstream research aimed at achieving industrial acceptability of the enzyme. Aquatic habitats of extreme temperatures are considered a potential metagenomic resource of heat-tolerant biocatalysts of industrial importance. The present study explored the thermal-spring metagenome of the Tattapani geothermal region, Chhattisgarh, India, discovering a novel d-allulose 3-epimerase gene, daeM, encoding an enzyme of high-level heat stability. The daeM gene was expressed in the microbial cells of a nonpathogenic and safe-grade species, B. subtilis, which was found to be capable of performing d-fructose to d-allulose interconversion via a whole-cell catalysis reaction. The results indicate that DaeM is a potential biocatalyst for commercial production of the rare sugar d-allulose. The study established that extreme environmental niches represent a genomic resource of functional sugar-related biocatalysts.

Don't Take It 'Lytely': A Case of Acute Tetany.

The most common causes of tetany are hypocalcemia, hypomagnesemia, hypokalemia, and alkalosis. Most case reports of tetany in the literature include some combination of the above metabolic derangements leading to non-life-threatening symptoms. We present a unique case of severe life-threatening tetany in a 38-year-old female with a history of Crohn's disease. She was previously dependent on total parenteral nutrition (TPN) but discontinued TPN two weeks prior to presentation due to the improvement of her Crohn's symptoms with a new medication regimen. We propose that malabsorption led to multiple electrolyte abnormalities, resulting in acute tetany that subsequently caused rhabdomyolysis. This case reviews the most common causes of acute tetany and highlights the interaction between electrolytes implicated in both tetany and rhabdomyolysis. It also emphasizes the importance of considering tetany as a diagnosis in a patient with unstable vital signs and diffuse muscle spasms.

Kinetic characterization of laccase from Bacillus atrophaeus, and its potential in juice clarification in free and immobilized forms.

In the present study, a laccase gene (BaLc) from a lignin degrading bacterium, Bacillus atrophaeus, has been cloned and expressed in Escherichia coli. The optimal catalytic activity of the protein was achieved at 5.5 pH and 35°C temperature, measured by oxidation of ABTS. The Km and Vmax values were determined as 1.42 mM and 4.16 µmole/min, respectively. To achieve the enzyme recovery, the biocatalyst (BaLc) was covalently attached onto the functionalized iron magnetic-nanoparticles. The nanoparticles were characterized by zeta-potential and FTIR analyses. The immobilized BaLc enzyme was physico-kinetically characterized, exhibiting retention of 60% of the residual activity after ten reaction cycles of ABTS oxidation. The immobilized biocatalyst system was tested for its biotechnological exploitability in plant juice processing, achieving 41-58% of phenol reduction, 41-58% decolorization, 50-59% turbidity reduction in the extracts of banana pseudo-stem and sweet sorghum stalk, and apple fruit juice. This is the first study to demonstrate the use of nanoparticle-laccase conjugate in juice clarification. The findings suggest that B. atrophaus laccase is a potential catalytic tool for plant juice bioprocessing activities.

Elevated Procalcitonin in Acute Pseudogout Flare: A Case Report.

An 86-year-old male presented to the emergency department with fevers and tachycardia. Given concern for sepsis, a broad infectious workup was pursued. Though no source of infection was identified, the patient was found to have an elevated procalcitonin level. The patient subsequently developed painful joint effusions of the knees, which on arthrocentesis were consistent with calcium pyrophosphate deposition (CPPD) or "pseudogout". His symptoms of fevers and arthralgias resolved with anti-inflammatory agents. CPPD is a known cause of systemic inflammatory response syndrome (SIRS) in elderly patients. Procalcitonin has been studied as a biomarker for distinguishing infectious from non-infectious causes of SIRS, although its use in CPPD and other crystal arthropathies is not well-defined. The current case is the first to describe elevated procalcitonin specifically attributable to acute pseudogout flare and highlights the need for further study of this biomarker in non-infectious, pro-inflammatory states.

A Case of Granulomatosis with Polyangiitis: Consequences of Delayed Diagnosis in a Life-threatening Malady.

Granulomatosis with polyangiitis (GPA) is one of three described anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Early diagnosis and treatment of GPA is paramount, as it may help prevent irreversible end-organ damage, especially renal and pulmonary failure. A 72-year-old male with a past medical history of lung adenocarcinoma in remission, chronic sinusitis status-post multiple sinus surgeries, and coronary artery disease presented with shortness of breath, dark urine, and asymmetric polyarthralgias. He had an acute kidney injury, leukocytosis, with urinalysis demonstrating pyuria and hematuria, without casts. Chest imaging showed cavitary nodular opacities in addition to interval increase of existing nodules compared to the most recent scan one month prior. His acute kidney injury progressed to renal failure requiring hemodialysis, and he developed an inflammatory polyarthritis. GPA was suspected clinically so he was started on high-dose intravenous corticosteroids, and subsequently plasmapheresis and rituximab. Serology returned with highly positive proteinase-3 antibodies, and cytoplasmic ANCA positivity on immunofluorescence. Renal biopsy demonstrated severely active pauci-immune glomerulonephritis. Several months after discharge, the patient passed away from gram positive bacteremia. This patient's recurrent sinusitis, pulmonary nodules, and subsequent renal failure were highly suggestive of GPA. A biopsy is recommended to confirm the diagnosis of GPA, but treatment should not be delayed if there is a high index of suspicion for the disease. Induction therapy with corticosteroids combined with rituximab or cyclophosphamide has significantly decreased the mortality of patients with GPA. Patients with GPA often have preceding history of nasopharyngeal and upper airway disease, and can present with fluctuating pulmonary infiltrates. Early recognition and treatment of patients with GPA can prevent life-threatening complications and reduce mortality.

An integrated bio-process for production of functional biomolecules utilizing raw and by-products from dairy and sugarcane industries.

The study investigated an integrated bioprocessing of raw and by-products from sugarcane and dairy industries for production of non-digestible prebiotic and functional ingredients. The low-priced feedstock, whey, molasses, table sugar, jaggery, etc., were subjected to transglucosylation reactions catalyzed by dextransucrase from Leuconostoc mesenteroides MTCC 10508. HPLC analysis approximated production of about 11-14 g L-1 trisaccharide i.e. 2-α-D-glucopyranosyl-lactose (4-galactosyl-kojibiose) from the feedstock prepared from table sugar, jaggery, cane molasses and liquid whey, containing about 30 g L-1 sucrose and lactose each. The trisaccharide was hydrolysed into the prebiotic disaccharide, kojibiose, by employing recombinant ß-galactosidase from Escherichia coli. The enzyme ß-galactosidase achieved about 90% conversion of 2-α-D-glucopyranosyl-lactose into kojibiose. The D-fructose generated by catalytic reactions of dextransucrase was targeted for catalytic transformation into rare sugar, D-allulose (or D-psicose), by treating the samples with Smt3-D-psicose 3-epimerase. The catalytic reactions resulted in the conversion of ~ 25% D-fructose to D-allulose. These bioactive compounds are known to exert a plethora of benefits to human health, and therefore, are preferred ingredients for making functional foods.

Development of a thermo-stable and recyclable magnetic nanobiocatalyst for bioprocessing of fruit processing residues and D-allulose synthesis.

The aim of the study was to covalently immobilize Smt3-D-psicose 3-epimerase onto functionalized iron oxide magnetic nanoparticles. After immobilization, Km of the immobilized enzyme increased, however, Vmax was nearly the same as that of its free form, indicating that immobilization has no detrimental effects on its catalytic output. The covalent immobilization caused a reduction in the deactivation rate constant (kd) values leading to 4-5 fold enhancement in its half-life at 50-65°C, indicating significant thermal stability of the iron-enzyme nanobioconjugate. The immobilized enzyme showed excellent storage stability by losing only 20% activity even after 60days of storage at 4°C. The immobilized enzyme retained up to 90% of its initial activity even after 10 consecutive cycles of catalyzing D-fructose epimerization reactions. Thus, after immobilization the enzyme exhibited remarkable improvements in thermal tolerance, storage stability and recycling efficiency, useful for development of industrially exploitable process for D-allulose production.

A novel approach of integrated bioprocessing of cane molasses for production of prebiotic and functional bioproducts.

In this work, the sugar industry by-product cane molasses was investigated as feedstock for acceptor reactions by dextransucrase from Leuconostoc mesenteroides MTCC 10508, leading to the biosynthesis of oligosaccharides. The starch industry corn fiber residue was used as a source for acceptor molecules, maltose, in the reaction. Production of approximately 124g oligosaccharides (DP3-DP6) per kg of fresh molasses was achieved. Further, cane molasses based medium was demonstrated as a sole carbon source for L. mesenteroides growth and dextransucrase production. d-Fructose released by dextransucrase activity as processing by-product was transformed into the functional monosaccharide with zero caloric value, d-psicose, by inducing its epimerization. Quantitative analysis approximated 37g d-psicose per kg of fresh molasses. Thus, the study established a novel approach of integrated bioprocessing of cane molasses into prebiotic and functional food additives.

Improved operational stability of d-psicose 3-epimerase by a novel protein engineering strategy, and d-psicose production from fruit and vegetable residues.

The aim of the present work was to improve stability of d-psicose 3-epimerase and biotransformation of fruit and vegetable residues for d-psicose production. The study established that N-terminal fusion of a yeast homolog of SUMO protein - Smt3 - can confer elevated optimal temperature and improved operational stability to d-psicose 3-epimerase. The Smt3-d-psicose 3-epimerase conjugate system exhibited relatively better catalytic efficiency, and improved productivity in terms of space-time yields of about 8.5kgL(-1)day(-1). It could serve as a promising catalytic tool for the pilot scale production of the functional sugar, d-psicose. Furthermore, a novel approach for economical production of d-psicose was developed by enzymatic and microbial bioprocessing of fruit and vegetable residues, aimed at epimerization of in situd-fructose to d-psicose. The bioprocessing led to achievement of d-psicose production to the extent of 25-35% conversion (w/w) of d-fructose contained in the sample.

Hyperbaric oxygen therapy for irradiated patients who require dental implants: a Cochrane review of randomised clinical trials.

OBJECTIVES: To compare success, morbidity, patient satisfaction and cost effectiveness of dental implant treatment carried out with and without hyperbaric oxygen (HBO) therapy in irradiated patients. DATA SOURCES: MEDLINE, EMBASE, The Cochrane Oral Health Group's Trials Register, and The Cochrane Central Register of Controlled Trials (CENTRAL) to June 2007. REVIEW METHODS: Systematic review with search without language restriction for randomised clinical trials only. Quality assessment of identified trials according to Cochrane Handbook for Systematic Reviews of Interventions. RESULTS: We identified one RCT measuring dental implant outcomes in patients who had received radiotherapy. Twenty-six patients were randomised to HBO therapy or no HBO therapy. There were no statistically significant differences by group for prosthesis or implant failure, preimplantation or postimplantation complication, or patient satisfaction. This trial had a high risk of bias. CONCLUSIONS: We were unable to find any strong evidence to either support or refute the use of HBO therapy for improving the implant outcome for patients who have received radiotherapy. We would recommend that more RCTs are undertaken to provide better evidence for decision making by clinicians. Meanwhile, clinicians should be aware of the limited availability of evidence and should therefore provide patients with the appropriate information about the potential benefits and harms of HBO therapy as part of the consent process.