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1.
Plants (Basel) ; 12(21)2023 Oct 25.
Article in English | MEDLINE | ID: mdl-37960035

ABSTRACT

The taxonomic assignment of sequences obtained by high throughput amplicon sequencing poses a limitation for various applications in the biomedical, environmental, and agricultural fields. Identifications are constrained by the length of the obtained sequences and the computational processes employed to efficiently assign taxonomy. Arriving at a consensus is often preferable to uncertain identification for ecological purposes. To address this issue, a new tool called "ASVmaker" has been developed to facilitate the creation of custom databases, thereby enhancing the precision of specific identifications. ASVmaker is specifically designed to generate reference databases for allocating amplicon sequencing data. It uses publicly available reference data and generates specific sequences derived from the primers used to create amplicon sequencing libraries. This versatile tool can complete taxonomic assignments performed with pre-trained classifiers from the SILVA and UNITE databases. Moreover, it enables the generation of comprehensive reference databases for specific genes in cases where no directly applicable database exists for taxonomic classification tools.

2.
Plant Dis ; 103(11): 2843-2850, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31469361

ABSTRACT

Cranberry fruit rot (CFR) is arguably one of the most limiting factors of cranberry (Vaccinium macrocarpon) production throughout its growing areas. The disease is caused by a group of closely related fungi that require identification using long and cumbersome steps of isolation and microscopic observations of structural features. The objective of this study was to develop a molecular assay to simultaneously detect and discriminate 12 of the most important fungal species reported to be pathogenic on cranberry fruit to facilitate the diagnosis of CFR. As the first approach, internal transcribed spacers and large subunit regions of all fungi were sequenced and confirmed with sequences available in the NCBI database. These data were used to develop primers able to differentiate seven of the 12 species. The five remaining species, including three in the Phacidiaceae family and two in the Glomerellaceae family, were differentiated on the basis of a more discriminant marker, the translation elongation factor 1-α. Two PCR reactions were optimized to clearly delineate the 12 species. The multiplex test was first validated using pure fungal cultures; it was subsequently validated using fruit collected in cranberry beds in eastern Canada. In the latter case, the test was rigorous enough to clearly discriminate the fungal pathogens from contaminants. Within the tested samples, Physalospora vaccinii and Coleophoma empetri were most commonly found. This molecular test offers scientists, diagnosticians, and growers a powerful tool that can rapidly and precisely identify fungi causing CFR so they can implement appropriate control methods.


Subject(s)
Ascomycota , Molecular Typing , Mycological Typing Techniques , Polymerase Chain Reaction , Vaccinium macrocarpon , Ascomycota/classification , Ascomycota/genetics , Canada , Fruit/microbiology , Molecular Typing/methods , Mycological Typing Techniques/methods , Reproducibility of Results , Vaccinium macrocarpon/microbiology
3.
Am J Physiol Cell Physiol ; 312(5): C550-C561, 2017 May 01.
Article in English | MEDLINE | ID: mdl-28179233

ABSTRACT

Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.


Subject(s)
Phosphates/metabolism , Silicon/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIb/chemistry , Sodium-Phosphate Cotransporter Proteins, Type IIb/metabolism , Sodium/metabolism , Animals , Female , Rats , Rats, Sprague-Dawley , Species Specificity
4.
J Plant Physiol ; 200: 82-9, 2016 Aug 01.
Article in English | MEDLINE | ID: mdl-27344403

ABSTRACT

Silicon (Si) is a beneficial element to plants, and its absorption via transporters leads to protective effects against biotic and abiotic stresses. In higher plants, two groups of root transporters for Si have been identified: influx transporters (Lsi1) and efflux transporters (Lsi2). Lsi1 transporters belong to the NIPIII aquaporins, and functional Lsi1s have been found in many plants species. Much less is known about Lsi2s that have been characterized in only a few species. Horsetail (Equisetum arvense), known among the highest Si accumulators in the plant kingdom, is a valuable model to study Si absorption and deposition. In this study, we first analyzed discrete Si deposition patterns in horsetail shoots, where ubiquitous silicification differs markedly from that of higher plants. Then, using the sequenced horsetail root transcriptome, two putative Si efflux transporter genes, EaLsi2-1 and EaLsi2-2, were identified. These genes share low sequence similarity with their homologues in higher plants. Further characterisation of EaLsi2-1 in transient expression assay using Nicotiana benthamiana epidermal cells confirmed transmembrane localization. In order to determine their functionality, the EaLsi2-1 was expressed in Xenopus oocytes, confirming that the translated protein was efficient for Si efflux. Both genes were equally expressed in roots and shoots, but interestingly, showed a much higher expression in the shoots than in the roots in contrast to Lsi2s found in other plants, a result consistent with the specific anatomy of horsetail and its rank as one of the highest Si accumulators among plant species.


Subject(s)
Equisetum/metabolism , Membrane Transport Proteins/metabolism , Plant Proteins/metabolism , Silicon/metabolism , Animals , Biological Transport , Cell Membrane/metabolism , Cloning, Molecular , DNA, Complementary/genetics , Equisetum/genetics , Genes, Plant , Membrane Transport Proteins/genetics , Oocytes/metabolism , Phylogeny , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Shoots/metabolism , Sequence Alignment , Xenopus
5.
Plant J ; 83(3): 489-500, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26095507

ABSTRACT

The controversy surrounding silicon (Si) benefits and essentiality in plants is exacerbated by the differential ability of species to absorb this element. This property is seemingly enhanced in species carrying specific nodulin 26-like intrinsic proteins (NIPs), a subclass of aquaporins. In this work, our aim was to characterize plant aquaporins to define the features that confer Si permeability. Through comparative analysis of 985 aquaporins in 25 species with differing abilities to absorb Si, we were able to predict 30 Si transporters and discovered that Si absorption is exclusively confined to species that possess NIP-III aquaporins with a GSGR selectivity filter and a precise distance of 108 amino acids (AA) between the asparagine-proline-alanine (NPA) domains. The latter feature is of particular significance since it had never been reported to be essential for Si selectivity. Functionality assessed in the Xenopus oocyte expression system showed that NIPs with 108 AA spacing exhibited Si permeability, while proteins differing in that distance did not. In subsequent functional studies, a Si transporter from poplar mutated into variants with 109- or 107-AA spacing failed to import, and a tomato NIP gene mutated from 109 to 108 AA exhibited a rare gain of function. These results provide a precise molecular basis to classify higher plants into Si accumulators or excluders.


Subject(s)
Aquaporins/genetics , Oligopeptides/genetics , Silicon/metabolism , Animals , Genomics , Xenopus laevis
6.
Mol Plant Pathol ; 16(6): 572-82, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25346281

ABSTRACT

On absorption by plants, silicon (Si) offers protection against many fungal pathogens, including powdery mildews. The mechanisms by which Si exerts its prophylactic role remain enigmatic, although a prevailing hypothesis suggests that Si positively influences priming. Attempts to decipher Si properties have been limited to plants able to absorb Si, which excludes the model plant Arabidopsis because it lacks Si influx transporters. In this work, we were able to engineer Arabidopsis plants with an Si transporter from wheat (TaLsi1) and to exploit mutants (pad4 and sid2) deficient in salicylic acid (SA)-dependent defence responses to study their phenotypic response and changes in defence expression against Golovinomyces cichoracearum (Gc) following Si treatment. Our results showed that TaLsi1 plants contained significantly more Si and were significantly more resistant to Gc infection than control plants when treated with Si, the first such demonstration in a plant transformed with a heterologous Si transporter. The resistant plants accumulated higher levels of SA and expressed higher levels of transcripts encoding defence genes, thus suggesting a role for Si in the process. However, TaLsi1 pad4 and TaLsi1 sid2 plants were also more resistant to Gc than were pad4 and sid2 plants following Si treatment. Analysis of the resistant phenotypes revealed a significantly reduced production of SA and expression of defence genes comparable with susceptible controls. These results indicate that Si contributes to Arabidopsis defence priming following pathogen infection, but highlight that Si will confer protection even when priming is altered. We conclude that Si-mediated protection involves mechanisms other than SA-dependent defence responses.


Subject(s)
Arabidopsis/immunology , Ascomycota/pathogenicity , Salicylic Acid/metabolism , Silicon/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Gene Expression Regulation, Plant , Mutation
7.
Plant Mol Biol ; 83(4-5): 303-15, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23771580

ABSTRACT

Silicon (Si) confers several benefits to many plant species when absorbed as silicic acid through nodulin 26-like intrinsic proteins (NIPs). The NIPs belong to major intrinsic protein (MIP) family, members of which form channels with high selectivity to control transport of water and different solutes. Here, comparative genomic analysis of the MIPs was performed to investigate the presence of Si transporter MIPs in soybean. Thorough analysis of phylogeny, gene organization, transcriptome profiling and protein modeling was performed to characterize MIPs in rice, Arabidopsis and soybean. Based on several attributes, two putative Si transporter genes, GmNIP2-1 and GmNIP2-2, were identified, characterized and cloned from soybean. Expression of both genes was detected in shoot and root tissues, and decreased as Si increased. The protein encoded by GmNIP2-2 showed functionality for Si transport when expressed in Xenopus oocytes, thus confirming the genetic capability of soybean to absorb the element. Comparative analysis of MIPs in plants provides opportunities to decipher gene evolution, functionality and selectivity of nutrient uptake mechanisms. Exploitation of this strategy has helped to uncover unique features of MIPs in soybean. The identification and functional characterization of Si transporters can be exploited to optimize the benefits that plants can derive from Si absorption.


Subject(s)
Aquaporins/genetics , Genome, Plant/genetics , Glycine max/genetics , Membrane Transport Proteins/genetics , Plant Proteins/genetics , Silicon/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Aquaporins/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Biological Transport , Conserved Sequence , Gene Expression Profiling , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Oryza/genetics , Oryza/metabolism , Phosphorylation , Phylogeny , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Plant Shoots/genetics , Plant Shoots/metabolism , Sequence Alignment , Silicon/analysis , Glycine max/metabolism , Transcriptome , Xenopus
8.
Plant Cell ; 24(7): 3119-34, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22805434

ABSTRACT

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Fruit/enzymology , Membrane Lipids/metabolism , Solanum lycopersicum/enzymology , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/isolation & purification , Down-Regulation/genetics , Fruit/chemistry , Fruit/genetics , Fruit/ultrastructure , Gene Expression Regulation, Plant/genetics , Gene Silencing , Solanum lycopersicum/chemistry , Solanum lycopersicum/genetics , Solanum lycopersicum/ultrastructure , Membrane Lipids/chemistry , Microscopy, Atomic Force , Plant Epidermis/chemistry , Plant Epidermis/enzymology , Plant Epidermis/genetics , Plant Epidermis/ultrastructure , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , Proteomics , RNA Interference , Waxes/chemistry , Waxes/metabolism
9.
Plant J ; 72(2): 320-30, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22712876

ABSTRACT

Plants benefit greatly from silicon (Si) absorption provided that they contain Si transporters. The latter have recently been identified in the roots of some higher plants known to accumulate high concentrations of Si, and all share a high level of sequence identity. In this study, we searched for transporters in the primitive vascular plant Equisetum arvense (horsetail), which is a valuable but neglected model plant for the study of Si absorption, as it has one of the highest Si concentrations in the plant kingdom. Our initial attempts to identify Si transporters based on sequence homology with transporters from higher plants proved unsuccessful, suggesting a divergent structure or property in horsetail transporters. Subsequently, through sequencing of the horsetail root transcriptome and a search using amino acid sequences conserved in plant aquaporins, we were able to identify a multigene family of aquaporin Si transporters. Comparison of known functional domains and phylogenetic analysis of sequences revealed that the horsetail proteins belong to a different group than higher-plant Si transporters. In particular, the newly identified proteins contain a STAR pore as opposed to the GSGR pore common to all previously identified Si transporters. In order to determine its functionality, the proteins were heterologously expressed in both Xenopus oocytes and Arabidopsis, and the results showed that the horsetail proteins are extremely efficient a transporting Si. These findings offer new insights into the elusive properties of Si and its absorption by plants.


Subject(s)
Aquaporins/genetics , Equisetum/genetics , Multigene Family , Silicon/metabolism , Amino Acid Sequence , Animals , Aquaporins/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Base Sequence , Biological Transport , Equisetum/metabolism , Female , Gene Expression , Models, Molecular , Molecular Sequence Data , Oocytes , Organ Specificity , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Plant Shoots/genetics , Plant Shoots/metabolism , Protein Structure, Tertiary , RNA, Plant/genetics , Sequence Alignment , Sequence Analysis, DNA , Transcriptome , Xenopus/genetics , Xenopus/metabolism
10.
Plant Mol Biol ; 79(1-2): 35-46, 2012 May.
Article in English | MEDLINE | ID: mdl-22351076

ABSTRACT

Silicon (Si) is known to be beneficial to plants, namely in alleviating biotic and abiotic stresses. The magnitude of such positive effects is associated with a plant's natural ability to absorb Si. Many grasses can accumulate as much as 10% on a dry weight basis while most dicots, including Arabidopsis, will accumulate less than 0.1%. In this report, we describe the cloning and functional characterization of TaLsi1, a wheat Si transporter gene. In addition, we developed a heterologous system for the study of Si uptake in plants by introducing TaLsi1 and OsLsi1, its ortholog in rice, into Arabidopsis, a species with a very low innate Si uptake capacity. When expressed constitutively under the control of the CaMV 35S promoter, both TaLsi1 and OsLsi1 were expressed in cells of roots and shoots. Such constitutive expression of TaLsi1 or OsLsi1 resulted in a fourfold to fivefold increase in Si accumulation in transformed plants compared to WT. However, this Si absorption caused deleterious symptoms. When the wheat transporter was expressed under the control of a root-specific promoter (a boron transporter gene (AtNIP5;1) promoter), a similar increase in Si absorption was noted but the plants did not exhibit symptoms and grew normally. These results demonstrate that TaLsi1 is indeed a functional Si transporter as its expression in Arabidopsis leads to increased Si uptake, but that this expression must be confined to root cells for healthy plant development. The availability of this heterologous expression system will facilitate further studies into the mechanisms and benefits of Si uptake.


Subject(s)
Genes, Plant/genetics , Membrane Transport Proteins/genetics , Plant Proteins/genetics , Silicon/metabolism , Triticum/genetics , Absorption/drug effects , Amino Acid Sequence , Animals , Arabidopsis/drug effects , Arabidopsis/genetics , Base Sequence , Cell Membrane/drug effects , Cell Membrane/metabolism , Cloning, Molecular , Gene Expression Regulation, Plant/drug effects , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Phenotype , Phylogeny , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Proteins/metabolism , Plant Roots/drug effects , Plant Roots/metabolism , Plant Shoots/drug effects , Plant Shoots/metabolism , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Protein Transport/drug effects , Sequence Alignment , Silicon/pharmacology , Nicotiana/drug effects , Nicotiana/metabolism , Triticum/drug effects , Triticum/growth & development , Triticum/metabolism , Xenopus laevis
11.
Plant Mol Biol ; 78(4-5): 323-36, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22170036

ABSTRACT

The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.


Subject(s)
Bryopsida/growth & development , Plant Proteins/metabolism , Plant Shoots/growth & development , RNA-Binding Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Molecular Sequence Data , Mutation , Phenotype , Phylogeny , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Shoots/metabolism , Poaceae/genetics , RNA-Binding Proteins/genetics
12.
Planta ; 231(3): 525-35, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19943172

ABSTRACT

TERMINAL EAR1-like (TEL) genes encode putative RNA-binding proteins only found in land plants. Previous studies suggested that they may regulate tissue and organ initiation in Poaceae. Two TEL genes were identified in both Populus trichocarpa and the hybrid aspen Populus tremula x P. alba, named, respectively, PoptrTEL1-2 and PtaTEL1-2. The analysis of the organisation around the PoptrTEL genes in the P. trichocarpa genome and the estimation of the synonymous substitution rate for PtaTEL1-2 genes indicate that the paralogous link between these two Populus TEL genes probably results from the Salicoid large-scale gene-duplication event. Phylogenetic analyses confirmed their orthology link with the other TEL genes. The expression pattern of both PtaTEL genes appeared to be restricted to the mother cells of the plant body: leaf founder cells, leaf primordia, axillary buds and root differentiating tissues, as well as to mother cells of vascular tissues. Most interestingly, PtaTEL1-2 transcripts were found in differentiating cells of secondary xylem and phloem, but probably not in the cambium itself. Taken together, these results indicate specific expression of the TEL genes in differentiating cells controlling tissue and organ development in Populus (and other Angiosperm species).


Subject(s)
Plant Proteins/genetics , Populus/genetics , Amino Acid Sequence , Cell Differentiation , Evolution, Molecular , Gene Duplication , Genome, Plant , In Situ Hybridization , Molecular Sequence Data , Phylogeny , Plant Proteins/chemistry , Plant Proteins/metabolism , Populus/growth & development , Populus/metabolism , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, Protein , Transcription, Genetic
13.
Gastroenterol Clin Biol ; 31(4): 431-5, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17483784

ABSTRACT

AIM: Acute pancreatitis is an important cause of morbidity and mortality, mainly due to sepsis. The aim of this study was to determine the incidence of infectious complications and their impact on mortality in patients hospitalized for acute pancreatitis. PATIENTS AND METHODS: Patients admitted for acute pancreatitis were retrospectively included within a period between 1995 and 2000. Incidence of abdominal and extra-abdominal sepsis and specific care were specifically analyzed. Risk factors for death were evaluated by uni- and multivariated analysis. RESULTS: Two hundreds and twelve consecutive patients (128 males, median age 54 years) were included. Mortality was 10.8%. At least one infectious episode was collected in 25% of the patients with an abdominal sepsis (26.8%), bacteriemia (24.4%), respiratory (24.4%) and urinary tracts (19.5%) infections. Infection was polymicrobial in 37.5%. An antibiotic prophylaxis was administered in 10.8%, more often in patients with severe pancreatitis. It did not alter mortality or incidence of infections but significantly delayed occurrence of sepsis. Mortality of patients treated with more than one line of antibiotics was higher. However in this study infectious complications were not an independent factor for mortality. CONCLUSION: Infections are frequent and polymicrobial but are not an independent prognostic factor during acute pancreatitis.


Subject(s)
Bacterial Infections/epidemiology , Pancreatitis/complications , APACHE , Acute Disease , Adult , Age Factors , Aged , Aged, 80 and over , Amoxicillin/administration & dosage , Amoxicillin/therapeutic use , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/therapeutic use , Antibiotic Prophylaxis , Azithromycin/administration & dosage , Azithromycin/therapeutic use , Bacterial Infections/drug therapy , Ceftriaxone/administration & dosage , Ceftriaxone/therapeutic use , Data Interpretation, Statistical , Drug Therapy, Combination , Female , Humans , Incidence , Male , Middle Aged , Pancreatitis/diagnosis , Pancreatitis/drug therapy , Pancreatitis/mortality , Pancreatitis/surgery , Prognosis , Retrospective Studies , Risk Factors , Sepsis/drug therapy , Sepsis/epidemiology , Time Factors
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