The utility of an under-distended gallbladder on ultrasound in ruling out acute cholecystitis.

PURPOSE: To study the association between gallbladder dimensions and acute cholecystitis and to define a sensitive cutoff for excluding the disease. MATERIALS AND METHODS: 456 consecutive patients with an abdominal ultrasound performed for right upper quadrant pain, from 1/2019 to 4/2019, were retrospectively collected. Length and width of the gallbladder were measured by a blinded radiology fellow. Patient charts were examined for prospective sonographic findings, clinical data, and pathology from subsequent cholecystectomy or at least 1 month of follow-up with symptom resolution. Univariable and multivariable logistic regression analysis were conducted to define the association of gallbladder dimensions and other sonographic and clinical variables with acute cholecystitis. Optimal and sensitive cutoffs of gallbladder widths were defined. The determined sensitive cutoff was validated with a separate cohort of 501 consecutive patients. RESULTS: 319 patients (median age 48 ± 19 years) including 11%, 19%, and 70% with acute, chronic, and no cholecystitis were included in the experimental cohort, respectively, and 298 patients (median age 50 ± 19 years) including 10%, 12%, and 79% with acute, chronic, and no cholecystitis were included in the validation cohort, respectively. Of all sonographic findings and clinical data, gallbladder width produced the best univariate ROC curve with an AUC of 0.91 (Odds ratio 5.1, 95% CI 3.1-8.5, p < 0.001). 2.2 cm was the gallbladder width cutoff below which there were no cases of acute cholecystitis in the experimental cohort. Multivariable logistic regression analysis using sonographic findings only produced an ROC curve with an AUC of 0.94. Applying the 2.2 cm cutoff in the validation cohort resulted in 100% sensitivity. CONCLUSION: Lack of gallbladder distention, defined as a width less than 2.2 cm, has potential to serve as a highly sensitive sign for exclusion of acute cholecystitis, regardless of additional sonographic findings and clinical data.

The Negative Predictive Value of a PI-RADS Version 2 Score of 1 on Prostate MRI and the Factors Associated With a False-Negative MRI Study.

OBJECTIVE. The purpose of this study was to calculate the negative predictive value of a prostate MRI study with a Prostate Imaging Reporting and Data System version 2 (PI-RADSv2) score of 1 (hereafter referred to as a PI-RADS 1 MRI study) and to explore the patient characteristics and MRI-based factors associated with an MRI study with false-negative results. MATERIALS AND METHODS. A total of 542 consecutive patients with a PI-RADS 1 MRI study obtained between January 2016 and July 2019 were retrospectively identified. Patient charts were examined to identify those patients who subsequently underwent systematic prostate biopsy within 1 year of undergoing MRI or at any later date if the biopsy was negative. Patient characteristics and MRI-specific factors were recorded. Two blinded radiologists evaluated the quality of the axial T2-weighted, DWI, and apparent diffusion coefficient sequences; measured the volume of the bladder, the prostate gland, and rectal gas; and determined whether the peripheral zone was avidly enhancing and whether low signal intensity was seen in 50% or more of the peripheral zone on T2-weighted images. Interobserver agreement was tested. Univariable and multivariable logistic regression models were built. RESULTS. A total of 150 patients (median age, 63 years; interquartile range, 56-70 years) were included. Of these patients, 19 (13%) had prostate cancer with a Gleason score of 3 + 4 or greater, yielding a negative predictive value of 87%. Both low T2 signal intensity in the peripheral zone and the prostate-specific antigen level were associated with a false-negative PI-RADS 1 assessment (odds ratio, 4.9 [95% CI, 1.6-14.9; p = 0.006] and 1.1 [95% CI, 1.0-1.2; p = 0.03], respectively). A cutoff prostate-specific antigen level of 3.97 ng/mL resulted in sensitivity and specificity of 89% and 21%, respectively. There was moderate interobserver agreement for low T2 signal intensity in the peripheral zone (κ coefficient = 0.75). CONCLUSION. Even among select patients who undergo subsequent biopsy because of a high clinical suspicion of prostate cancer, a PI-RADS 1 prostate MRI study has a high negative predictive value. A T2-hypointense peripheral zone and an elevated prostate-specific antigen level are significantly associated with a false-negative MRI study.

The fucosidase-pool of Emticicia oligotrophica: Biochemical characterization and transfucosylation potential.

Three novel bacterial α-l-fucosidases, which cleave terminal fucosyl residues from glycoconjugates are reported in this work. Originating from the recently discovered bacterium Emticicia oligotrophica, recombinant fucosidase isoforms designated as Eo0918, Eo3066 and Eo3812 were shown to have the highest activity between pH 6.0 and 7.0 and temperature optima between 30 and 45°C. All enzymes catalyzed the hydrolysis of the model substrate pNP-α-l-fucose and revealed significantly different regiospecificities towards fucose-containing oligosaccharides: Eo0918 liberated exclusively α1,6-linked fucose and Eo3812 released only α1,3-fucosyl residues, whereas Eo3066 showed broader substrate promiscuity. The enzymatic activity of Eo0918 and Eo3812 increased upon the addition of Ca(2+), Mn(2+) and Zn(2+) ions, whereas the activity of Eo3066 was significantly decreased in the presence of these metal ions. In addition, Eo0918 also catalyzed the transfer of fucose from pNP-α-l-fucose to the 7-hydroxyl group of 4-methylumbelliferone with up to 15% transglycosylation yield. Facile recombinant expression in E. coli, distinct substrate specificities and the transglycosylation ability of Eo0918 presented herein make these newly discovered fucosidases valuable candidates for bioanalytical and biotechnological applications.

Rapid Sample Preparation Methodology for Plant N-Glycan Analysis Using Acid-Stable PNGase H+.

The quantification of potentially allergenic carbohydrate motifs of plant and insect glycoproteins is increasingly important in biotechnological and agricultural applications as a result of the use of insect cell-based expression systems and transgenic plants. The need to analyze N-glycan moieties in a highly parallel manner inspired us to develop a quick N-glycan analysis method based on a recently discovered bacterial protein N-glycanase (PNGase H(+)). In contrast to the traditionally used PNGase A, which is isolated from almond seeds and only releases N-glycans from proteolytically derived glycopeptides, the herein implemented PNGase H(+) allows for the release of N-glycans directly from the glycoprotein samples. Because PNGase H(+) is highly active under acidic conditions, the consecutive fluorescence labeling step using 2-aminobenzamide (2AB) can be directly performed in the same mixture used for the enzymatic deglycosylation step. All sample handling and incubation steps can be performed in less than 4 h and are compatible with microwell-plate sampling, without the need for tedious centrifugation, precipitation, or sample-transfer steps. The versatility of this methodology was evaluated by analyzing glycoproteins derived from various plant sources using ultra-performance liquid chromatography (UPLC) analysis and further demonstrated through the activity analysis of four PNGase H(+) mutant variants.

Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila.

Since the isolation and identification of Akkermansia muciniphila one decade ago, much attention has been drawn to this gut bacterium due to its role in obesity and type 2 diabetes. This report describes the discovery and biochemical characterisation of all four putative neuraminidases annotated in the A. muciniphila genome. Recombinantly expressed candidate genes, which were designated Am0705, Am0707, Am1757 and Am2085, were shown to cover complementary pH ranges between 4.0 and 9.5. Temperature optima of the enzymes lay between 37 and 42 °C. All four enzymes were strongly inhibited by Cu(2+) and Zn(2+), and loss of activity after the addition of EDTA suggests that all neuraminidases, with the exception of Am0707, require divalent metal ions for their catalytic function. Chemoenzymatically synthesised α2,3- and α2,6-linked indoyl-sialosides were utilised to determine the regioselectivity and substrate promiscuity of the neuraminidases towards C5-modifications of sialic acids with N-acetyl-, N-glycolyl-, N-propionyl-, or hydroxyl-groups. The combination of simple purification procedures and good activities of some of the characterised neuraminidases makes them potentially interesting as tools in bioanalytical or industrial applications.

Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus.

UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus, a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60 °C, with almost no loss of activity after 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn(2+) ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose.

Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A.

Peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H+. The gene of PNGase H+ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H+ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H+ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.