Martin S. Keller

Lignin content in natural Populus variants affects sugar release

The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plants recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production.

Exploring nitrilase sequence space for enantioselective catalysis.

ABSTRACT Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 106 to 1010 members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.

Targeted Access to the Genomes of Low-Abundance Organisms in Complex Microbial Communities

ABSTRACT Current metagenomic approaches to the study of complex microbial consortia provide a glimpse into the community metabolism and occasionally allow genomic assemblies for the most abundant organisms. However, little information is gained for the members of the community present at low frequencies, especially those representing yet-uncultured taxa, which include the bulk of the diversity present in most environments. Here we used phylogenetically directed cell separation by fluorescence in situ hybridization and flow cytometry, followed by amplification and sequencing of a fraction of the genomic DNA of several bacterial cells that belong to the TM7 phylum. Partial genomic assembly allowed, for the first time, a look into the evolution and potential metabolism of a soil representative from this group of organisms for which there are no species in stable laboratory cultures. Genomic reconstruction from targeted cells of uncultured organisms isolated directly from the environment represents a powerful approach to access any specific members of a community and an alternative way to assess the communitys metabolic potential.

Early fixation reduces morbidity and mortality in elderly patients with hip fractures from low-impact falls

OBJECTIVE To determine the effect of the timing of fracture fixation and the physiologic status on admission of elderly patients with hip fractures from low impact falls on resource utilization and outcome. METHODS A 5-year retrospective review of 82 elderly (age > 65 years) patients with isolated low-impact hip fractures stratified into early (< 24 hours), intermediate (24 to 72 hours), and late (> 72 hours) operative fixation. Admission Acute Physiology and Chronic Health Evaluation (APACHE) II scores, number of comorbidities, fracture type, complication rate, length of stay, discharge acuity, and mortality were calculated for each group. RESULTS Values are mean +/- SD. The mean admission APACHE II score of the entire group was 8.1 +/- 0.2, indicating that these patients were physiologically stable on arrival. The mean numbers of comorbidities or APACHE II were not significant between groups. No differences existed in the mean APACHE II scores for survivors and nonsurvivors (7.95 +/- 2.34 vs. 9.17 +/- 3.06, p = 0.2409). There were no differences in the mean APACHE II scores and predicted survival for each group. However, a significant decrease in actual survival was observed with late fixation (p < 0.001; Fishers Exact Test). Patients who were fixed late also had a significantly higher infectious morbidity (p = 0.00469), length of stays (p = 0.0226), and total hospital cost (p = 0.0001), compared with those fixed early or immediate, despite having no difference in average acuity upon discharge (p = 0.3883). CONCLUSIONS Delay in fracture fixation, in elderly patients who are physiologically stable on admission, significantly increases morbidity and mortality and adversely affects resource utilization.

Acute ovarian torsion in children

BACKGROUND Acute ovarian torsion (OT) is an uncommon cause of abdominal pain in children and is frequently confused with other conditions. METHODS We reviewed the records (1983 to 1999) of all children treated for acute OT at our childrens hospital. RESULTS Mean child age (n = 51) was 12.5 +/- 0.3 years. Children presented with either right-sided (n = 29) or left-sided (n = 22) pain. Diagnosis of OT was confirmed preoperatively by ultrasound (73%) or computed tomography (CT) scan (10%) while nine children (17%) with right-sided pain underwent surgery for presumed appendicitis. Despite a relatively short time from diagnosis to surgery, all 51 children required salpingooophorectomy. Contralateral biopsy was performed in 29% and 57% had an appendectomy. Younger children more commonly had either a mature cystic teratoma or torsion with no underlying abnormality as an etiology compared with OT in older children that was more likely to result from either a follicular or corpus luteal cyst. Pathologic examination of the contralateral ovary and appendix was normal in all children who underwent biopsy and appendectomy. CONCLUSION Ultrasonography with color doppler is helpful for differentiating acute OT from appendicitis. Although the twisted ovary can rarely be salvaged, the etiology is usually benign. Preoperative serum markers and contralateral ovary biopsy may be unnecessary.

Unusual Microbial Xylanases from Insect Guts

ABSTRACT Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted β-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Environmental Proteomics: a Paradigm Shift in Characterizing Microbial Activities at the Molecular Level

SUMMARY The increase in sequencing capacity led to a new wave of metagenomic projects, enabling and setting the prerequisite for the application of environmental proteomics technologies. This review describes the current status of environmental proteomics. It describes sample preparation as well as the two major technologies applied within this field: two-dimensional electrophoresis-based environmental proteomics and liquid chromatography-mass spectrometry-based environmental proteomics. It also highlights current publications and describes major scientific findings. The review closes with a discussion of critical improvements in the area of integrating experimental mass spectrometry technologies with bioinformatics as well as improved sample handling.

Blunt renal trauma in children: Experience with conservative management at a pediatric trauma center

BACKGROUND The authors reviewed the outcome for children with blunt renal injury managed with a nonoperative protocol at their pediatric trauma center. METHODS Fifty-five consecutive children aged 0.5 to 17 years with blunt renal injury managed over a 14-year period were reviewed. All patients were evaluated with computed tomographic scanning. Injuries were graded according to the American Association for the Surgery of Trauma Organ Injury Scale. RESULTS Forty-eight of 55 children (87%) were successfully managed nonoperatively. Overall, there were 5 grade I, 13 grade II, 18 grade III, 14 grade IV, and 5 grade V injuries. All children with grades I and III injuries were successfully managed nonoperatively. Two (6%) of these children required transfusion. Only four (29%) children with grade IV and three (60%) with grade V injuries required surgical interventions (one nephrostomy, six nephrectomies). Excluding patients with continuing hemorrhage, only 2 (14%) of 14 with high-grade injuries required surgical intervention (1 nephrostomy, 1 nephrectomy). Clearance of gross hematuria correlated with severity of injury and was prolonged in grade IV and V compared with grade I to III injuries (6.8 +/- 2.7 vs. 3.2 +/- 2.1 days, respectively; p < 0.05). Fifty-one children (93%) available for follow-up were normotensive with normal renal function. CONCLUSION These data support the use of conservative management for all grades in stable children with blunt renal injury. Transfusion requirements, operative rates, and outcome are consistent with other pediatric solid organ injuries.

Conservative management of pancreatic trauma in children.

Many adults and most children with a solid-organ abdominal injury can be managed nonoperatively. To date, however, little is known about the outcome of nonoperative management of pancreatic injury. To analyze current treatment patterns of pancreatic injury in children, all children (age < 19 years) identified in the National Pediatric Trauma Registry (49,540 patients) and admitted to two level I pediatric trauma centers with a diagnosis of injury to the pancreas (International Classification of Disease-9 codes 863.81-863.84 and 863.91-863.94) were reviewed. Over a 7-year period, 154 children were identified with pancreatic injury. Thirty-one (20%) sustained severe injuries (grades III, IV, or V) and 123 (80%) sustained lower-grade injuries (grades I and II). Sixteen (52%) of the children sustaining grades III, IV, or V injury required pancreatic procedures (9 distal resections, 3 simple repairs, 2 enteric anastomoses, 2 others). Only 26 (21%) of the grades I and II injuries required surgical intervention specific to the pancreas (11 resections, 9 catheter drainage of pseudocysts, 2 enteric anastomoses, 4 others). Ninety-seven (79%) grades I and II injuries were successfully managed conservatively. Overall, 15 (10%) children required drainage procedures for pseudocyst. The frequency of operative intervention decreased during the last 4 years of the study (18 vs. 26%, p > 0.05), coinciding with a decrease in the frequency of drainage procedures for pseudocysts. The need for surgical intervention was not influenced by age, Injury Severity Score, or Pediatric Trauma Score (p > 0.05). Associated abdominal injuries were common but did not influence operations on the pancreas (p > 0.05). No deaths were attributed to the pancreatic injury. These data indicate that early intervention for pancreatic injury, in the absence of clinical deterioration or major ductal injury (grades III, IV, or V), is unwarranted, and careful observation may supplant the conventional surgical therapy recommended for adults.

High‐Throughput Cultivation of Microorganisms Using Microcapsules

This chapter describes a universal and novel method that provides access to the immense reservoir of untapped microbial diversity by cultivation. This technique uses microcapsules to encapsulate single cells combined with parallel microbial cultivation under low nutrient flux conditions. Under these conditions, single encapsulated cells grow and form microcolonies within the microcapsules. Flow cytometry is used as a sensitive tool to detect growth within the microcapsules. Microcapsules that contain microcolonies (originated from a single encapsulated cell) are sorted individually into microtiter dishes containing organic-rich medium. This high-throughput cultivation can provide more than 10,000 bacterial and fungal isolates per environmental sample.