Progression from biochemical leak to pancreatic fistula after distal pancreatectomy. Don't cry over spilt amylase.

BACKGROUND: Postoperative pancreatic fistula (POPF) is a frequent complication after distal pancreatectomy (DP), but its upgrading from biochemical leak (BL) still represents an unexplored phenomenon. This study aims at identifying risk factors of the clinical evolution from BL to grade-B POPF after DP. METHODS: Patients who underwent DP between 2015 and 2019 and who developed either BL (n = 89,56%) or BL upgraded to late B fistula (LB) after postoperative day 5 (n = 71,44%) were included. Preoperative, surgical, postoperative predictors were compared between the two groups. RESULTS: Patients with LB were significantly older (61 vs 56 years, P < 0.025) and received neoadjuvant chemotherapy more frequently (22.5% vs 8.5%,P = 0.017). Extended lymphadenectomy (52.8% vs 31.0%,P = 0.006), longer operative times (OT) (307 vs 250 min,P = 0.002), greater estimated blood loss (250 vs 150 ml, P = 0.021), and the appearance of purulent fluid in surgical drains (58.4% vs 21.1%; P < 0.001) were more frequently observed in LB group. Only purulent fluid in surgical drains and longer OT were confirmed as independent predictors of BL clinical progression. CONCLUSIONS: Purulent fluid from surgical drains should be suspicious of BL upgrading. Frail patients undergoing longer interventions may represent key targets of mitigation strategies to minimize the magnitude of an incipient fistula and its increase in morbidity.

Sporadic non-functioning pancreatic neuroendocrine tumours: multicentre analysis.

BACKGROUND: Outcomes after surgery for sporadic pancreatic neuroendocrine neoplasms (Pan-NENs) were evaluated. METHODS: This multicentre study included patients who underwent radical pancreatic resection for sporadic non-functioning Pan-NENs. In survival analysis, the risk of mortality in this cohort was analysed in relation to that of the matched healthy Italian population. Relative survival (RS) was calculated as the rate between observed and expected survival. Factors related to RS were investigated using multivariable modelling. RESULTS: Among 964 patients who had pancreatic resection for sporadic non-functioning Pan-NENs, the overall RS rate was 91.8 (95 per cent c.i. 81.5 to 96.5) per cent. 2019 WHO grade (hazard ratio (HR) 5.75 (s.e. 4.63); P = 0.030) and European Neuroendocrine Tumour Society (ENETS) TNM stage (6.73 (3.61); P < 0.001) were independent predictors of RS. The probability of a normal lifespan for patients with G1, G2, G3 Pan-NENS, and pancreatic neuroendocrine carcinomas (Pan-NECs) was 96.7, 54.8, 0, and 0 per cent respectively. The probability of a normal lifespan was 99.8, 99.3, 79.8, and 46.8 per cent for those with stage I, II, III, and IV disease respectively. The overall disease-free RS rate was 73.6 (65.2 to 79.5) per cent. 2019 WHO grade (HR 2.10 (0.19); P < 0.001) and ENETS TNM stage (HR 2.50 (0.24); P < 0.001) significantly influenced disease-free RS. The probability of disease-free survival was 93.2, 84.9, 45.2, and 6.8 per cent for patients with stage I, II, III, and IV disease, and 91.9, 45.2, 9.4, and 0.7 per cent for those with G1, G2, G3 Pan-NENS, and Pan-NECs, respectively. CONCLUSION: A surgical approach seems without benefit for Pan-NECs, and unnecessary for small G1 sporadic Pan-NENs. Surgery alone may be insufficient for stage III-IV and G3 Pan-NENs.

Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation.

The DNA in the core of spores of Bacillus species is saturated with a group of small, acid-soluble proteins (SASP) that protect DNA from a variety of harsh treatments and play a major role in spore resistance and long-term spore survival. During spore germination, SASPs are rapidly degraded to amino acids and this degradation is initiated by a sequence-specific protease called germination protease (GPR), which exhibits no obvious mechanistic or amino acid sequence similarity to any known class of proteases. GPR is synthesized during sporulation as an inactive tetrameric zymogen termed P(46), which later autoprocesses to a smaller form termed P(41), which is active only during spore germination. Here, we report the crystal structure of P(46) from Bacillus megaterium at 3.0 A resolution and the fact that P(46) monomer adopts a novel fold. The asymmetric unit contains two P(46) monomers and the functional tetramer is a dimer of dimers, with an approximately 9 A channel in the center of the tetramer. Analysis of the P(46) structure and site-directed mutagenesis studies have provided some insight into the mechanism of zymogen activation as well as the zymogen's lack of activity and the inactivity of P(41) in the mature spore.

Crystallization and preliminary diffraction studies of a truncated form of a novel protease from spores of Bacillus megaterium.

During germination of spores of Bacillus species, a novel protease termed GPR initiates the degradation of a group of small acid-soluble spore proteins which protect the dormant spore's DNA from damage. Trypsin digestion of the zymogen of B. megaterium GPR removes approximately 15 kDa from the C-terminal end of the 46 kDa zymogen subunit, leaving a 30 kDa subunit. Single crystals of this truncated form of GPR have been obtained by the vapor-diffusion method using PEG 4000 as a precipitating agent. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 67.99, b = 105.34, c = 108.63 A, beta = 95.68 degrees. The cryofrozen crystals diffract X-rays to about 3.3 A using synchrotron radiation.

Structural studies of a novel germination protease from spores of Bacillus megaterium.

The amino acid sequence-specific protease (termed GPR) in the bacterium Bacillus megaterium initiates the rapid degradation of small, acid-soluble spore proteins during the germination of spores of this organism. GPR is synthesized during spore formation as an inactive zymogen termed P46, which later autoprocesses to a smaller active form termed P41, which acts during spore germination. However, GPR exhibits no obvious mechanistic or amino acid sequence similarity to any of the known classes of proteases. To initiate the determination of the mechanisms of P46 to P41 conversion, P46 inactivity, and P41 catalysis, B. megaterium GPR has been overexpressed in Escherichia coli and purified to homogeneity by anion-exchange and size exclusion chromatography, and crystals of both P46 and P41 have been obtained by the vapor diffusion method. P46 crystals diffracted x rays to 3.5 A but the crystals of P41 diffracted x rays to only 6.5 A. A native x-ray diffraction data set of P46 has been collected; the unit cell parameters are a = b = 76.8, c = 313.1 A, alpha = beta = gamma = 90 degrees; the space group is tetragonal P41212 or P43212. The asymmetric unit contains two monomeric molecules with a crystal volume per unit protein mass of 2. 85 A3/Da and a solvent content of about 57%. An isomorphous heavy atom derivative data set has also been obtained for P46 crystals with potassium dicyanoaurate (I).

Structure and mechanism of action of the protease that degrades small, acid-soluble spore proteins during germination of spores of Bacillus species.

The germination protease (GPR) of Bacillus megaterium initiates the degradation of small, acid-soluble proteins during spore germination. Trypsin treatment of the 46-kDa GPR zymogen (termed P46) removes an approximately 15-kDa C-terminal domain generating a 30-kDa species (P30) which is stable against further digestion. While P30 is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P46 to the active 41-kDa form of GPR (P41). Trypsin treatment of P41 cleaves the same bond in the C-terminal part of the protein as is cleaved in the P46-->P30 conversion. While the approximately 29-kDa species generated by trypsin treatment of P41 is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P41 which is 13 degreesC lower than that for P46 and the unfolding of P41 at significantly lower concentrations of guanidine hydrochloride than for P46, are further evidence for a difference in tertiary structure between P46 and P41, with P46 presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P46 and P41. The removal of approximately 30% of P46 or P41 without significant loss in enzyme activity localized GPR's catalytic residues to the N-terminal two-thirds of the molecule. This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P41 by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.

Most of the propeptide is dispensable for stability and autoprocessing of the zymogen of the germination protease of spores of Bacillus species.

Loss of 3, 7, or 10 of the amino-terminal 15 residues removed upon autoactivation of the zymogen of the germination protease (GPR), which initiates protein degradation during germination of spores of Bacillus species, did not result in significant changes in (i) the lack of enzymatic activity of the zymogen, (ii) the rate of zymogen autoactivation, or (iii) the unreactivity of the zymogen's single SH group. Removal of 13 amino-terminal residues resulted in a partially active enzyme whose SH group was as reactive as the fully active enzyme. These findings suggest that at least a part of the propeptide blocks access to the enzyme's active site. However, the free propeptide did not inhibit the enzyme.

Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis.

NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. The three-dimensional structure of NH3-dependent NAD+ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X-ray crystallography (at 2.6 and 2.0 angstroms resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques. The enzyme consists of a tight homodimer with alpha/beta subunit topology. The catalytic site is located at the parallel beta-sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg2+ ion are observed. Residue Ser46, part of the neighboring 'P-loop', is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido-NAD+ during the first step of the catalyzed reaction. The deamido-NAD+ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center. A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine-dependent GMP synthetase, supporting the hypothesis that NAD+ synthetase belongs to the newly discovered family of 'N-type' ATP pyrophosphatases.

Crystallization of NAD+ synthetase from Bacillus subtilis.

NAD(+)-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD+. Mutants of NAD+ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD+ synthetase, an enzyme fundamental for NAD+ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD+-->NAD+ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria.

Role of FlgM in sigma D-dependent gene expression in Bacillus subtilis.

The alternative sigma factor sigma D directs transcription of a number of genes involved in chemotaxis, motility, and autolysis in Bacillus subtilis (sigmaD regulon). The activity of SigD is probably in contrast to that of FlgM, which acts as an antisigma factor and is responsible for the coupling of late flagellar gene expression to the assembly of the hook-basal body complex. We have characterized the effects of an in-frame deletion mutation of flgM. By transcriptional fusions to lacZ, we have shown that in FlgM-depleted strains there is a 10-fold increase in transcription from three different sigmaD-dependent promoters, i.e., Phag, PmotAB, and PfliDST. The number of flagellar filaments was only slightly increased by the flgM mutation. Overexpression of FlgM from a multicopy plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible spac promoter drastically reduced the level of transcription from the hag promoter. On the basis of these results, we conclude that, as in Salmonella typhimurium, FlgM inhibits the activity of SigD, but an additional element is involved in determining the number of flagellar filaments.

The outB gene of Bacillus subtilis codes for NAD synthetase.

The outB gene of Bacillus subtilis is involved in spore germination and outgrowth and is essential for growth. The OutB protein was obtained by expression in Escherichia coli and purified to apparent homogeneity. Here we report experiments showing that OutB is a NH3-dependent NAD synthetase, the enzyme that catalyzes the final reaction in the biosynthesis of NAD. The enzyme is composed of two identical subunits of 30,240 Da and is NH3-dependent, whereas glutamine is inefficient as an amide donor. The NAD synthetase is highly resistant to heat, with a half-time of inactivation at 100 degrees C of 13 min. A mutant NAD synthetase was purified from a B. subtilis strain temperature-sensitive during spore germination and outgrowth. The mutant enzyme was 200 times less active than the wild-type one, with a lower temperature optimum and a non-hyperbolic kinetic versus NH4+. The time course of synthesis of OutB showed that synthesis of the enzyme started during germination and outgrowth, and reached the highest level at the end of exponential growth. The enzyme could be recovered from dormant spores.