bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature Dependent Viscoplastic Simulation of Controlled Collapse Solder Joint Under Thermal Cycling
Authors: Sarihan V.
Published: April 2008

Solder is being extensively used in electronic packages for both electrical and mechanical connection. Solder joints are subjected to severe operating conditions and hence their reliability is very critical for the packages. Simulation is very effective for understanding, predicting and design improvement of electronic packages where solder is the prime joiner, however all the material response complexities of solder over the temperature regime it is subjected to should be modelled. Solder in electronic components very often operates at 0.6 to 0.8 times it’s melting point. In this regime the time dependent material response (creep and stress relaxation) is very significant and can no longer be ignored. Also the plastic and creep responses of solder alloys have a very strong dependence on temperature. A nonlinear finite element based methodology has been developed for simulation of solder alloy over its full regime of material behavior, also accounting for the strong temperature dependence. This includes elastic, time independent plastic, and time dependent viscoplastic response. The methodology has been used for predicting the response of a flip chip with 95Pb5 percent Sn peripheral bumps subjected to thermal cycling. Correlation is observed between the location of failure in the bump and the maximum inelastic strain. The importance of doing a multicycle simulation is demonstrated and a direction is indicated for the bump size modification for flip chip bump design improvements for greater reliability.
Analysis and Simulation of Thermal Transients and Resultant Stresses and Strains in TAB Packaging
Authors: Jog M. A. , Cohen I. M. , Ayyaswamy P. S.
Published: April 2008

During normal power cycling of the electronic equipment, the differing coefficients of thermal expansion result in differential elongations. Because each level of packaging is subject to mounting constraints, the differential strains result in bending and shear stresses. Repeated duty cycling can cause fatigue at joints, at interfaces between different materials, at interconnection locations, or cause delamination of composite materials. Accelerated Thermal Cycling (ATC) is done to simulate the fatigue failures that may arise because of this power cycling. The current practice is to determine ATC stresses by assuming that the temperatures of various layers are equal and constant. In this study, we have relaxed the isothermal assumption and we provide results for thermal stresses and strains in a first level package. This is accomplished by accurately determining the transient temperature fields in various layers of the package. Temperature variations for different heat transfer coefficients have also been calculated. The results indicate that realistic estimates of thermal stresses and strains are only possible with models that allow for temperature variation in the body of the package. High equivalent stress values are obtained at the chip-heat sink interface and in the bumps connecting the leads to the chip.
A Detailed Thermal Model of the Infrared Reflow Soldering Process
Authors: Eftychiou M. A. , Bergman T. L. , Masada G. Y.
Published: April 2008

A thermal model of the infrared reflow soldering process is developed and sample predictions are reported. The model, which accounts for coupled radiation, mixed convection and conduction heat transfer, is capable of predicting relevant thermal effects ranging from the convection characteristics within the IR oven to the detailed thermal response, including solid-liquid phase change, of individual solder connections. Although the heat transfer here is radiatively dominated, mixed convection and conduction effects are important in determining the thermal response of the card assembly.
Physical and Fuzzy Logic Modeling of a Flip-Chip Thermocompression Bonding Process
Authors: Kang Sa-Yoon , Xie H. , Lee Y. C.
Published: April 2008

Flip-Chip connections using gold-to-gold, gold-to-aluminum, or gold-to-solder bondings or contacts enhanced by epoxy are low-cost alternatives to soldering. To assist their technology advancements, we have developed yield models for a representative assembly process with flip-chip, thermocompression bondings. Based on bonding mechanics, a physical yield model has been developed to characterize the process. Then, a fuzzy logic model has been established to improve the modeling’s accuracy by including experimental data. The physical yield model can predict the assembly yield as a function of forces and planarities of the end effector, bump height variations, bump geometries, mechanical properties corresponding to different materials and temperatures, and distribution patterns of bumps. Consistent with our experimental experience, the calculated force level for a high-yield process was around 3000 gmf for a 30-gold-bump chip with a bump diameter of 60 μm and a height of 50 μm. The fuzzy logic model can be trained and adjusted by the results of physical models and experiments. It correlates very well to the nonlinear relationships between the yield and the assembly parameters, and has a self-learning capability to update itself with new data. Such capabilities have been demonstrated by studying the bonding on a substrate with or without a compliant layer.
Optimization of Enhanced Surfaces for High Flux Chip Cooling by Pool Boiling
Authors: Mudawar I. , Anderson T. M.
Published: April 2008

A high flux electronic chip was numerically and experimentally simulated to investigate pool boiling capabilities of enhanced metallic surface attachments built upon a 12.7 × 12.7 mm2 base area. It is shown how experimental nucleate boiling data for a flat chip and for chips with low-profile microstructures can be used as input boundary conditions in the numerical prediction of boiling performances of high flux, smooth and microstructured extended cylindrical surfaces. A technique for extending the applicability of the numerical results to cylindrical fin arrays is demonstrated with the aid of experimental data obtained for these surfaces. Surface enhancement resulted in chip planform heat fluxes of 105.4 and 159.3 W/cm2, for saturated and 35°C subcooled FC-72, respectively.
Thermal Stress Measurement in Silicon Chips Encapsulated in IC Plastic Packages Under Temperature Cycling
Authors: Miura Hideo , Kitano Makoto , Nishimura Asao , Kawai Sueo
Published: April 2008

Thermal stress in silicon chips encapsulated in IC plastic packages is discussed. A stress-sensing chip which can detect the three-dimensional stress components separately is developed. Thermal stress occurs in the silicon chip almost linearly with temperature at the steady state. The stress path with temperature during heating is the same as that during cooling. On the other hand, during temperature cycling, stress hysteresis is observed, and the stress increases gradually with increasing cycles. The stress increase phenomenon occurs due to the viscoelastic phenomenon of the molding compounds. The stress-increase ratio is determined by the relationship between the cycle period and the stress relaxation time of the molding compounds at temperatures of interest.
Constitutive Behavior and Low Cycle Thermal Fatigue of 97Sn-3Cu Solder Joints
Authors: Pao Y.-H. , Badgley S. , Jih E. , Govila R. , Browning J.
Published: April 2008

The thermal cyclic shear stress/strain hysteresis response and associated steady-state creep parameters of 97Sn-3Cu solder joints have been determined using a beam specimen previously developed by Pao et al. (1992a). The solder joint was subjected to a 40-minute thermal cycling from 40°C to 140°C. A constitutive equation based on elastic and steady-state creep deformation for the solder has been formulated and implemented in a finite element program, ABAQUS, to model the experiment. The results show that the constitutive equation based on one single creep mechanism cannot fully account for the deformation during cooling, as opposed to the case of 90Pb-10Sn where the entire cyclic deformation can be well modeled by a similar constitutive equation (Pao et al., 1992c). This suggests that another creep mechanism is dominant for lower stresses and higher temperature. The thermal fatigue results show that the failure mechanism of 97Sn-3Cu joints is similar to that of 90Pb-10Sn joints, but the number of cycles to failure of 97Sn-Cu solder joints is at least 5 times longer than 90Pb-10Sn solder joints. This indicates the potential application of 97Sn-3Cu in place of 90Pb-10Sn solder.
Fatigue Crack Growth Rate in 63Sn37Pb Solder Joints
Authors: Guo Z. , Conrad H.
Published: April 2008

Fatigue crack growth rate (FCGR) was determined in shear (Mode II) on notched 63Sn37Pb solder joints at room temperature. The FCGR data correlated reasonably well with the expression da/dN = B (ΔY)b where ΔY was either ΔK, ΔJ, or ΔWp. The value of b for ΔJ and ΔWp was similar to that generally obtained for metals in Mode I loading; however, it was 2-3 times larger in the case of ΔK. Although the fatigue cracks initiated at the centrally located notches, they soon wandered toward the solder/Cu interface and then propagated in the solder adjacent to the intermetallic compound. The fatigue crack mode was transgranular with no clear microstructure preference. The results obtained on the notched specimens are in accord with those reported previously on initially smooth specimens tested in shear.
The Influence of the Cycle Frequency and Wave Shape on the Fatigue Life of Leaded Chip Carrier Printed Wiring Board Interconnections
Authors: Solomon H. D.
Published: April 2008

This study employed different cycle frequencies, to determine the influence of cycle frequency on the fatigue life of leaded chip carrier printed wiring board (LCC/PWB) interconnections. Real LCC/PWB interconnections were mechanically cycled at 35°C or 125°C. The cycle frequency was varied by varying the ramp loading and unloading rates or by introducing hold times. Tests were run with equal hold times at the maximum and minimum displacements or only at the maximum displacement. The use of slower ramp cycling or the introduction in the hold times increased the plastic displacement and this decreased the fatigue life. This plastic displacement increase was corrected for, to unveil the underlying material response. The corrected data was similar to that observed in previous tests on simple, single, solder joints, where the plastic strain was kept constant while the cycle frequency was reduced. At 125°C there was a small influence of wave shape, with maximum displacement hold time tests showing the shortest fatigue lives and symmetric (maximum and minimum) hold time tests showing the longest fatigue lives. The fatigue lives of the tests run with ramp cycling were intermediate. This wave shape effect is relatively small, especially compared to the large overall influence of the cycle frequency.
Advances in Bonding Technology for Electronic Packaging
Authors: Lee Chin C. , Wang Chen Y. , Matijasevic Goran
Published: April 2008

Recent progress in bonding materials is briefly reviewed with highlights of some of the advantages and disadvantages of the various attachment processes. The principle and experimental results of bonding with multilayer structures of Au-Sn and Au-In are presented. Using solid state as well as liquid phase diffusion of the multilayers, bonding temperatures less than the final melting point of the alloy can be used. This technique therefore allows reversal of the conventional soldering step hierarchy allowing a higher temperature process to follow the multilayer bonding step. Proper deposition of the multilayers inhibits oxidation of tin or indium. Die attachment experiments confirmed that high quality bonding can be obtained as seen in the void-free bonding layer images done by scanning acoustic microscopy. Cross-section examinations with SEM and EDX show near-eutectic alloy formation of good uniformity. Thermal shock tests confirmed the high strength of these solder alloys.
Prediction of Electrical Properties of Plain-Weave Fabric Composites for Printed Wiring Board Design
Authors: Agarwal R. K. , Dasgupta A.
Published: April 2008

A mechanistic model is presented for predicting the effective dielectric constant and loss tangent of woven-fabric reinforced composites with low-loss constituents. A two-scale asymptotic homogenization scheme is used to predict the orthotropic effective properties. A three-dimensional unit-cell enclosing the characteristic periodic repeat pattern in the fabric weave is isolated and modeled mathematically. Electrostatic boundary value problems (BVP’s) are formulated in the unit-cell and are solved analytically to predict effective dielectric constant of the composite, using three-dimensional series-parallel reactance nets. Results are also verified numerically, using finite element methods. The effective dielectric constant and the effective loss tangent are then obtained, analogous to mechanical viscoelastic problems for low-loss materials. The predicted dielectric constant and loss tangent are compared with experimental results for E-glass/epoxy laminates. Frequency dependence of the effective dielectric constant and loss tangent is obtained from the corresponding behavior of the constituent materials. Trade-off studies are conducted to investigate the effect of the constituent material properties on orthotropic effective dielectric permittivity.
Coating of Electronic Components by the RTV Dispersion—Part I: Physical Model of the Deposition Process Determined by Drop Tests
Authors: Owczarek J. A.
Published: April 2008

This paper describes a study of the process of deposition of RTV dispersion on electronic components placed on substrates. The objective was to develop a technique for the consistent manufacture of encapsulant coating of a desired thickness and extent. In addition, it was desired to obtain an understanding of the phenomenon of run-over, or wicking, of the RTV dispersion onto external leads of circuits being encapsulated, and of means to control it. In this paper physical properties of the RTV dispersion which influence the deposition process were determined using a novel drop test method. These properties allow building of a physical model of the deposition process, and its analysis. The results of drop tests show that the RTV dispersion behaves like a plastic “false body” material which possesses yield stress after a long rest, and which retains residual yield stress after shearing. Part I of this paper is concerned with building of the physical model of the encapsulant deposition process. It also deals with the derivation of an equation relating the wall shear stress to the encapsulant volumetric flow rate.
Time-Dependent Fracture of Polyimide Films
Authors: Popelar S. F. , Popelar C. H. , Kenner V. H.
Published: April 2008

A fracture mechanics approach for quantifying slow crack growth in thin polyimide films and assessing their structural integrity and life expectancy is presented. The methodology and techniques developed in this investigation may also be applied to other polymeric materials. A test protocol for studying slow crack growth is described. Room temperature fracture tests were performed and an analysis model was developed and validated to analyze the fracture tests. Correlations between the rate of crack growth and the crack driving force as measured by the stress intensity factor were made and contrasted for Kapton 100HN, 300H and 500HN polyimide films. The crack growth rate was found to depend very strongly upon the stress intensity factor. The practical implication of this finding is that the fracture of these polyimide films may be approximated as being controlled by a critical value of the stress intensity factor.
Planning of Component Placement/Insertion Sequence and Feeder Setup in PCB Assembly Using Genetic Algorithm
Authors: Leu M. C. , Wong H. , Ji Z.
Published: April 2008

A new application of the genetic algorithm approach is introduced to solve printed circuit board assembly planning problems. The developed genetic algorithm finds the sequence of component placement/insertion and the arrangement of feeders simultaneously, for achieving the shortest assembly time, for three main types of assembly machines. The algorithm uses links (parents) to represent possible solutions and it applies genetic operators to generate new links (offspring) in an iterative procedure to obtain nearly optimal solutions. Examples are provided to illustrate solutions generated by the algorithm.
SGI2, a Relative ofSalmonellaGenomic Island SGI1 with an Independent Origin
Authors: Levings Renee S. , Djordjevic Steven P. , Hall Ruth M.
Published: April 2008

Multiply antibiotic-resistantSalmonella entericaserovar Emek strains isolated in Australia and the United Kingdom had similar features, suggesting that they all belong to a single clone. These strains all contain SGI2 (formerly SGI1-J), an independently formed relative ofSalmonellagenomic island SGI1. In SGI2, the complex class 1 integron which includes all of the resistance genes is not located betweentnpR(S027) and S044 as in SGI1 and SGI1 variants. Instead,tnpRwas found to be adjacent to S044, and the integron is located 6.9 kb away, within S023. In both SGI1 and SGI2, the 25-bp inverted repeats that mark the outer ends of class 1 integrons are flanked by a 5-bp duplication of the target, indicating that incorporation of the integron was by transposition. A small number of differences between the sequences of the backbones of SGI1 and SGI2 were also found. Hence, a class 1 integron has entered two different variants of the SGI backbone to generate two distinct lineages. Despite this, the integron in SGI2 has a complex structure that is very similar to that of In104 in SGI1. Differences are in the cassette arrays and in the gene which encodes the chloramphenicol and florfenicol efflux protein. The CmlA9 protein, encoded by InEmek, is only 92.8% identical to FloRc (also a CmlA family protein) from SGI1. A variant form of SGI2, SGI2-A, which has lost thetet(G) andcmlA9resistance determinants, was found in one strain.
Acquisition of a Plasmid-Borne
bla
OXA-58
Gene with an Upstream IS
1008
Insertion Conferring a High Level of Carbapenem Resistance to
Acinetobacter baumannii
Authors: Chen Te-Li , Wu Roy Chen-Chih , Shaio Men-Fang , Fung Chang-Phone , Cho Wen-Long
Published: April 2008

The oxacillinase gene was reported to confer limited resistance to carbapenem in
Acinetobacter baumannii
. In this study, we have demonstrated that an
A. baumannii
clinical isolate harboring a plasmid, pTVICU53, has 11,037 bp encoding 13 open reading frames. A
bla
OXA-58
gene with an upstream insertion of truncated IS
Aba3
(ΔIS
Aba3
) and IS
1008
was found in this plasmid. ΔIS
Aba3
and IS
1008
provided two independent promoters for the transcription control of the
bla
OXA-58
gene. The transformation of pTVICU53 or a shuttle vector bearing IS
1008
-ΔIS
Aba3
-
bla
OXA-58
to different
A. baumannii
recipients can increase their MICs of carbapenem 64- to 256-fold. The deletion of promoters provided by IS
1008
resulted in dramatic decreases in
bla
OXA-58
transcription and a 32- to 64-fold reduction in the carbapenem MIC. These findings highlight that
A. baumannii
might develop carbapenem resistance with a single transformation step, taking up a plasmid containing a genetic construct with a potentially high level of transcription of the
bla
OXA-58
gene.
Functional Diversity among Metallo-β-Lactamases: Characterization of the CAR-1 Enzyme of
Erwinia carotovora
Authors: Stoczko Magdalena , Frère Jean-Marie , Rossolini Gian Maria , Docquier Jean-Denis
Published: April 2008

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and a lack of sensitivity to commercially available β-lactamase inactivators. Apart from the acquired subclass B1 enzymes, which exhibit increasing clinical importance and whose evolutionary origin remains unclear, most MBLs are encoded by resident genes found in the genomes of organisms belonging to at least three distinct phyla. Using genome database mining, we identified an open reading frame (ORF) (ECA2849) encoding an MBL-like protein in the sequenced genome of
Erwinia carotovora
, an important plant pathogen. Although no detectable β-lactamase activity could be found in
E. carotovora
, a recombinant
Escherichia coli
strain in which the ECA2849 ORF was cloned showed decreased susceptibility to several β-lactams, while carbapenem MICs were surprisingly poorly affected. The enzyme, named CAR-1, was purified by means of ion-exchange chromatography steps, and its characterization revealed unique structural and functional features. This new MBL was able to efficiently hydrolyze cephalothin, cefuroxime, and cefotaxime and, to a lesser extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized. CAR-1 is the first example of a functional naturally occurring MBL in the family
Enterobacteriaceae
(order
Enterobacteriales
) and highlights the extraordinary structural and functional diversity exhibited by MBLs.
Pharmacokinetics and Tissue Distribution of Anidulafungin in Rats
Authors: Damle Bharat , Stogniew Martin , Dowell James
Published: April 2008

This study assessed the tissue distribution of anidulafungin in rats. Anidulafungin rapidly distributed into tissues, achieving peak concentrations within 30 min, and maintained levels above MICs for common pathogens over 72 h. In tissues susceptible to fungal infection (liver, lung, spleen, kidney), exposure was 9- to 12-fold higher than in plasma.
Mutational Events in Cefotaximase Extended-Spectrum β-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance
Authors: Novais Ângela , Cantón Rafael , Coque Teresa M. , Moya Andrés , Baquero Fernando , Galán Juan Carlos
Published: April 2008

CTX-M β-lactamases, which show a high cefotaxime hydrolytic activity, constitute the most prevalent extended-spectrum β-lactamase (ESBL) type found among clinical isolates. The recent explosive diversification of CTX-M enzymes seems to have taken place due to the appearance of more efficient enzymes which are capable of hydrolyzing both cefotaxime and ceftazidime, especially among the CTX-M-1 cluster. A combined strategy of in vitro stepwise evolution experiments using
bla
CTX-M-1
,
bla
CTX-M-3
, and
bla
CTX-M-10
genes and site-directed mutagenesis has been used to evaluate the role of ceftazidime and other β-lactam antibiotics in triggering the diversity found among enzymes belonging to this cluster. Two types of mutants, P167S and D240G, displaying high ceftazidime MICs but reduced resistance to cefotaxime and/or cefepime, respectively, were identified. Such an antagonistic pleiotropic effect was particularly evident with P167S/T mutations. The incompatibility between P167S and D240G changes was demonstrated, since double mutants reduced susceptibility to both ceftazidime and cefotaxime-cefepime; this may explain the absence of strains containing both mutations in the clinical environment. The role of A77V and N106S mutations, which are frequently associated with P167S/T and/or D240G, respectively, in natural strains, was investigated. The presence of A77V and N106S contributes to restore a high-level cefotaxime resistance phenotype, but only when associated with mutations P167S and D240G, respectively. However, A77V mutation increases resistance to both cefotaxime and ceftazidime when associated with CTX-M-10. This suggests that in this context this mutation might be considered a primary site involved in resistance to broad-spectrum cephalosporins.
Tools for Functional Postgenomic Analysis ofListeria monocytogenes
Authors: Monk Ian R. , Gahan Cormac G. M. , Hill Colin
Published: April 2008

We describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations, and improved plasmid transfer by electroporation in the food-borne pathogenListeria monocytogenes. pIMK, a kanamycin-resistant, site-specific, integrative listeriophage vector was constructed and then modified for overexpression (pIMK2) or for isopropyl-β-d-thiogalactopyranoside (IPTG)-regulated expression (pIMK3 and pIMK4). The dynamic range of promoters was assessed by determining luciferase activity, P60 secretion, and internalin A-mediated invasion. These analyses demonstrated that pIMK4 and pIMK3 have a stringently controlled dynamic range of 540-fold. Stable gene overexpression was achieved with pIMK2, giving a range of expression for the three vectors of 1,350-fold. The lactococcal pORI280 system was optimized for the generation of chromosomal mutations and used to create five newprfAstar mutants. The combination of pIMK4 and pORI280 allowed streamlined creation of “IPTG-dependent” mutants. This was exemplified by creation of a clean deletion mutant with deletion of the universally essentialsecAgene, and this mutant exhibited a rapid loss of viability upon withdrawal of IPTG. We also improved plasmid transfer by electroporation into three commonly used laboratory strains ofL. monocytogenes. A 125-fold increase in transformation efficiency for EGDe compared with the widely used protocol of Park and Stewart (S. F. Park and G. S. Stewart, Gene 94:129-132, 1990) was observed. Maximal transformation efficiencies of 5.7 × 106and 6.7 × 106CFU per μg were achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 × 107CFU per μg is the highest efficiency reported thus far forL. monocytogenesF2365.
Molecular Analysis of the
Enterobacter sakazakii
O-Antigen Gene Locus
Authors: Mullane N. , O'Gaora P. , Nally J. E. , Iversen C. , Whyte P. , Wall P. G. , Fanning S.
Published: April 2008

Nucleotide polymorphism associated with the O-antigen-encoding locus,
rfb
, in
Enterobacter sakazakii
was determined by PCR-restriction fragment length polymorphism analysis. Based on the analysis of these DNA profiles, 12 unique banding patterns were detected among a collection of 62 strains from diverse origins. Two common profiles were identified and were designated serotypes O:1 and O:2. DNA sequencing of the 12,500-bp region flanked by
galF
and
gnd
identified 11 open reading frames, all with the same transcriptional direction. Analysis of the proximal region of both sequences demonstrated remarkable heterogeneity. A PCR assay targeting genes specific for the two prominent serotypes was developed and applied for the identification of these strains recovered from food, environmental, and clinical samples.
Coexistence of Antibiotic-Producing and Antibiotic-Sensitive Bacteria in Biofilms Is Mediated by Resistant Bacteria
Authors: Narisawa Naoki , Haruta Shin , Arai Hiroyuki , Ishii Masaharu , Igarashi Yasuo
Published: April 2008

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus
Brevibacillus
from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer,
Pseudomonas aeruginosa
strain P1. In addition, pyocyanin-resistant strains belonging to the genus
Raoultella
were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1,
Brevibacillus
strain S1, and
Raoultella
strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.
Effect of Extraction and Assay Media on Analysis of Airborne Endotoxin
Authors: Spaan Suzanne , Doekes Gert , Heederik Dick , Thorne Peter S. , Wouters Inge M.
Published: April 2008

The measurement of airborne endotoxins is thus far not standardized. Earlier studies reported higher endotoxin yields when Tween 20 was added to the media used for filter extraction and in the
Limulus
amebocyte lysate (LAL) assay. This study compared four common media and assessed the effects of Tween during extraction and analysis separately. Parallel airborne dust samples from five work environments (
n
= 250) were used to compare the four media (pyrogen-free water [PFW], PFW-Tween 20, PFW-Tris, and PFW-triethylamine-phosphate [TAP]) and an extraction time of 10 or 60 min. A subset of the extracts in PFW or PFW-Tween (
n
= 40) were analyzed in parallel LAL assays with PFW or PFW-Tween as the assay medium. The results produced by a shorter extraction time or the presence of Tris were similar to the results for the reference procedure (PFW and 60 min of shaking). The use of PFW-TAP showed overall lower yields and a deviant calibration curve. The presence of Tween in the extraction medium resulted in significantly (
P
< 0.05) higher endotoxin yields from all dust types, independent of the effect of Tween in the assay. Tween in the LAL assay, however, also strongly inhibited the reactivity of the lipopolysaccharide (LPS) standard, thus shifting the calibration curve to higher values. The inhibition of LPS in test samples was less pronounced and varied between dust sources, resulting in enhanced calculated concentrations. This assay effect could be circumvented by diluting extracts at least 50-fold before the LAL assay. In conclusion, of the media tested, only Tween enhances the efficiency of endotoxin extraction from airborne dust samples in a consistent manner. We recommend extraction in PFW-Tween combined with dilution and LAL analysis in PFW.
Loss of Virulence Genes in
Escherichia coli
Populations during Manure Storage on a Commercial Swine Farm
Authors: Duriez Patrick , Zhang Yun , Lu Zexun , Scott Andrew , Topp Edward
Published: April 2008

Confined livestock production farms typically store their wastes prior to land application. Here, we employed three complementary approaches to evaluate changes in the population structure and stability of virulence genes in
Escherichia coli
during manure storage on a commercial farm that housed healthy swine. Isolates were genotyped by repetitive extragenic palindromic PCR using the BOXA1R primer and evaluated for the presence of selected virulence genes by PCR. Isolates obtained from the manure holding tank (
n
= 392) carried
estB
,
fedA
,
stx
2e
,
astA
,
paa
,
aida-I
, and
sepA
at lower frequencies than isolates obtained from fresh feces (
n
= 412). Fresh fecal material from the barn was added into diffusion chambers and immersed in the manure holding tank for 7 weeks. The fecal
E. coli
population was initially dominated by a single genotype, all isolates of which carried
fedA
and
aida-I
. After 7 weeks, a genotype that did not carry any virulence genes dominated the surviving population. In a second experiment, 48 fecal isolates of
E. coli
that varied in their genotypes and virulence gene complement were incubated in diffusion chambers in the manure holding tank for 3 weeks. Over 95% of the inoculum population carried at least one virulence gene, whereas after 3 weeks 90% of the recovered isolates carried no virulence genes. Taken together, these results indicate that during commercial manure storage, there was a significant reduction in the carriage of these virulence genes by
E. coli
. We propose that loss of virulence genes from enteric pathogens in the farm and in natural environments may, if generalized, contribute to the attenuation of a public health risk from contamination with agricultural wastes.
Mycelium Differentiation and Antibiotic Production in Submerged Cultures ofStreptomyces coelicolor
Authors: Manteca Angel , Alvarez Ruben , Salazar Nuria , Yagüe Paula , Sanchez Jesus
Published: April 2008

Despite the fact that most industrial processes for secondary metabolite production are performed with submerged cultures, a reliable developmental model forStreptomycesunder these culture conditions is lacking. With the exception of a few species which sporulate under these conditions, it is assumed that no morphological differentiation processes take place. In this work, we describe new developmental features ofStreptomyces coelicolorA3(2) grown in liquid cultures and integrate them into a developmental model analogous to the one previously described for surface cultures. Spores germinate as a compartmentalized mycelium (first mycelium). These young compartmentalized hyphae start to form pellets which grow in a radial pattern. Death processes take place in the center of the pellets, followed by growth arrest. A new multinucleated mycelium with sporadic septa (second mycelium) develops inside the pellets and along the periphery, giving rise to a second growth phase. Undecylprodigiosin and actinorhodin antibiotics are produced by this second mycelium but not by the first one. Cell density dictates how the culture will behave in terms of differentiation processes and antibiotic production. When diluted inocula are used, the growth arrest phase, emergence of a second mycelium, and antibiotic production are delayed. Moreover, pellets are less abundant and have larger diameters than in dense cultures. This work is the first to report on the relationship between differentiation processes and secondary metabolite production in submergedStreptomycescultures.
Defects in Conidiophore Development and Conidium-Macrophage Interactions in a Dioxygenase Mutant of
Aspergillus fumigatus
Authors: Dagenais Taylor R. T. , Chung DaWoon , Giles Steven S. , Hull Christina M. , Andes David , Keller Nancy P.
Published: April 2008

Oxygenated fatty acids, or oxylipins, play an essential role in physiological signaling and developmental processes in animals, plants, and fungi. Previous characterization of three
Aspergillus fumigatus
dioxygenases (PpoA, PpoB, and PpoC), similar in sequence to mammalian cyclooxygenases, showed that PpoA is responsible for the production of the oxylipins 8
R
-hydroperoxyoctadecadienoic acid and 5
S
,8
R
-dihydroxy-9
Z
,12
Z
-octadecadienoic acid and that PpoC is responsible for 10
R
-hydroxy-8
E
,12
Z
-hydroperoxyoctadecadienoic acid. Here, Δ
ppo
mutants were characterized to elucidate the role of fungal dioxygenases in
A. fumigatus
development and host interactions. The Δ
ppoC
strain displayed distinct phenotypes compared to those of other Δ
ppo
mutants and the wild type, including altered conidium size, germination, and tolerance to oxidative stress as well as increased uptake and killing by primary alveolar macrophages. These experiments implicate oxylipins in pathogen development and suggest that Δ
ppoC
represents a useful model for studying the
A. fumigatus
-host interaction.
A Cyanobacterial Lipopolysaccharide Antagonist Inhibits Cytokine Production Induced byNeisseria meningitidisin a Human Whole-Blood Model of Septicemia
Authors: Jemmett Kim , Macagno Annalisa , Molteni Monica , Heckels John E. , Rossetti Carlo , Christodoulides Myron
Published: April 2008

Septicemia caused byNeisseria meningitidisis characterized by increasing levels of meningococcal lipopolysaccharide (Nm-LPS) and cytokine production in the blood. We have used an in vitro human whole-blood model of meningococcal septicemia to investigate the potential of CyP, a selective Toll-like receptor 4 (TLR4)-MD-2 antagonist derived from the cyanobacteriumOscillatoria planktothrixFP1, for reducing LPS-mediated cytokine production. CyP (≥1 μg/ml) inhibited the secretion of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6 (by >90%) and chemokines IL-8 and monocyte chemoattractant protein 1 (by ∼50%) induced by the treatment of blood with pure Nm-LPS, by isolated outer membranes, and after infection with live meningococci of different serogroups. In vitro studies with human dendritic cells and TLR4-transfected Jurkat cells demonstrated that CyP competitively inhibited Nm-LPS interactions with TLR4 and subsequent NF-κB activation. These data demonstrate that CyP is a potent antagonist of meningococcal LPS and could be considered a new adjunctive therapy for treating septicemia.
New Adhesin of Enteroaggregative
Escherichia coli
Related to the Afa/Dr/AAF Family
Authors: Boisen Nadia , Struve Carsten , Scheutz Flemming , Krogfelt Karen A. , Nataro James P.
Published: April 2008

Enteroaggregative
Escherichia coli
(EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized Tn
phoA
mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a Tn
phoA
insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the
aggR
gene. Cloning of the complete Hda gene cluster and expression in
E. coli
DH5α resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.
Effect of Multiple Genetic Polymorphisms on Antigen Presentation and Susceptibility toMycobacterium tuberculosisInfection
Authors: Chang Stewart T. , Linderman Jennifer J. , Kirschner Denise E.
Published: April 2008

Several molecules related to antigen presentation, including gamma interferon (IFN-γ) and the major histocompatibility complex (MHC), are encoded by polymorphic genes. Some polymorphisms were found to affect susceptibility to tuberculosis (TB) when they were considered singly in epidemiological studies, but how multiple polymorphisms interact to determine susceptibility to TB in an individual remains an open question. We hypothesized that polymorphisms in some genes may counteract or intensify the effects of polymorphisms in other genes. For example, an increase in IFN-γ expression may counteract the weak binding that a particular MHC variant displays for a peptide fromMycobacterium tuberculosisto establish the same T-cell response as another, more strongly binding MHC variant. To test this hypothesis, we developed a mathematical model of antigen presentation based on experimental data for the known effects of genetic polymorphisms and simulated time courses when multiple polymorphisms were present. We found that polymorphisms in different genes could affect antigen presentation to the same extent and therefore compensate for each other. Furthermore, we defined the conditions under which such relationships could exist. For example, increased IFN-γ expression compensated for decreased peptide-MHC affinity in the model only above a certain threshold of expression. Below this threshold, changes in IFN-γ expression were ineffectual compared to changes in peptide-MHC affinity. The finding that polymorphisms exhibit such relationships could explain discrepancies in the epidemiological literature, where some polymorphisms have been inconsistently associated with susceptibility to TB. Furthermore, the model allows polymorphisms to be ranked by effect, providing a new tool for designing association studies.
The Hybrid Sensor Kinase RscS Integrates Positive and Negative Signals To Modulate Biofilm Formation in
Vibrio fischeri
Authors: Geszvain Kati , Visick Karen L.
Published: April 2008

Overexpression of the
Vibrio fischeri
sensor kinase RscS induces expression of the
syp
(
sy
mbiosis
p
olysaccharide) gene cluster and promotes biofilm phenotypes such as wrinkled colony morphology, pellicle formation, and surface adherence. RscS is predicted to be a hybrid sensor kinase with a histidine kinase/ATPase (HATPase) domain, a receiver (Rec) domain, and a histidine phosphotransferase (Hpt) domain. Bioinformatic analysis also revealed the following three potential signal detection domains within RscS: two transmembrane helices forming a transmembrane region (TMR), a large periplasmic (PP) domain, and a cytoplasmic PAS domain. In this work, we genetically dissected the contributions of these domains to RscS function. Substitutions within the carboxy-terminal domain supported identification of RscS as a hybrid sensor kinase; disruption of both the HATPase and Rec domains eliminated induction of
syp
transcription, wrinkled colony morphology, pellicle formation, and surface adherence, while disruption of Hpt resulted in decreased activity. The PAS domain was also critical for RscS activity; substitutions in PAS resulted in a loss of activity. Generation of a cytoplasmic, N-terminal deletion derivative of RscS resulted in a partial loss of activity, suggesting a role for localization to the membrane and/or sequences within the TMR and PP domain. Finally, substitutions within the first transmembrane helix of the TMR and deletions within the PP domain both resulted in increased activity. Thus, RscS integrates both inhibitory and stimulatory signals from the environment to regulate biofilm formation by
V. fischeri
.
Spontaneous Amplification of the Actinorhodin Gene Cluster in
Streptomyces coelicolor
Involving Native Insertion Sequence IS
466
Authors: Widenbrant E. M. , Tsai Hsiu-Hui , Chen Carton W. , Kao C. M.
Published: April 2008

We observed a spontaneous amplification of the
Streptomyces coelicolor
chromosome, including genes encoding biosynthetic enzymes of the antibiotic actinorhodin. A new junction of two tandem segments has, inserted within it, a third copy of a transposable element existing in two places elsewhere in the chromosome, suggesting its involvement in the amplification mechanism.
Response Regulator DegU ofListeria monocytogenesControls Temperature-Responsive Flagellar Gene Expression in Its Unphosphorylated State
Authors: Mauder Norman , Williams Tatjana , Fritsch Frederike , Kuhn Michael , Beier Dagmar
Published: April 2008

We demonstrate that inListeria monocytogenes, temperature-responsive transcriptional control of flagellar genes does not rely on the phosphorylation of the conserved phosphorylation site (D55) in the receiver domain of response regulator DegU. Furthermore, proper control of DegU-regulated genes involved in ethanol tolerance and virulence is independent of receiver phosphorylation.
Salmonella enterica
Requires ApbC Function for Growth on Tricarballylate: Evidence of Functional Redundancy between ApbC and IscU
Authors: Boyd Jeffrey M. , Lewis Jeffrey A. , Escalante-Semerena Jorge C. , Downs Diana M.
Published: April 2008

Mutants of
Salmonella enterica
lacking
apbC
have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. Here we show that
apbC
is required for
S. enterica
to use tricarballylate as a carbon and energy source. Tricarballylate catabolism requires three gene products, TcuA, TcuB, and TcuC. Of relevance to this work is the TcuB protein, which has two [4Fe-4S] clusters required for function, making it a logical target for the
apbC
effect. TcuB activity was 100-fold lower in an
apbC
mutant than in the isogenic
apbC
+
strain. Genetic data show that derepression of the
iscRSUA-hscAB-fdx-orf3
operon or overexpression of
iscU
from a plasmid compensates for the lack of ApbC during growth on tricarballylate. The studies described herein provide evidence that the scaffold protein IscU has a functional overlap with ApbC and that ApbC function is involved in the synthesis of active TcuB.
The Genome of
Heliobacterium modesticaldum
, a Phototrophic Representative of the
Firmicutes
Containing the Simplest Photosynthetic Apparatus
Authors: Sattley W. Matthew , Madigan Michael T. , Swingley Wesley D. , Cheung Patricia C. , Clocksin Kate M. , Conrad Amber L. , Dejesa Liza C. , Honchak Barbara M. , Jung Deborah O. , Karbach Lauren E. , Kurdoglu Ahmet , Lahiri Surobhi , Mastrian Stephen D. , Page Lawrence E. , Taylor Heather L. , Wang Zi T. , Raymond Jason , Chen Min , Blankenship Robert E. , Touchman Jeffrey W.
Published: April 2008

Despite the fact that heliobacteria are the only phototrophic representatives of the bacterial phylum
Firmicutes
, genomic analyses of these organisms have yet to be reported. Here we describe the complete sequence and analysis of the genome of
Heliobacterium modesticaldum
, a thermophilic species belonging to this unique group of phototrophs. The genome is a single 3.1-Mb circular chromosome containing 3,138 open reading frames. As suspected from physiological studies of heliobacteria that have failed to show photoautotrophic growth, genes encoding enzymes for known autotrophic pathways in other phototrophic organisms, including ribulose bisphosphate carboxylase (Calvin cycle), citrate lyase (reverse citric acid cycle), and malyl coenzyme A lyase (3-hydroxypropionate pathway), are not present in the
H. modesticaldum
genome. Thus, heliobacteria appear to be the only known anaerobic anoxygenic phototrophs that are not capable of autotrophy. Although for some cellular activities, such as nitrogen fixation, there is a full complement of genes in
H. modesticaldum
, other processes, including carbon metabolism and endosporulation, are more genetically streamlined than they are in most other low-G+C gram-positive bacteria. Moreover, several genes encoding photosynthetic functions in phototrophic purple bacteria are not present in the heliobacteria. In contrast to the nutritional flexibility of many anoxygenic phototrophs, the complete genome sequence of
H. modesticaldum
reveals an organism with a notable degree of metabolic specialization and genomic reduction.