bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of HIF‐1 inhibitors for cancer therapy
Authors: Onnis Barbara , Rapisarda Annamaria , Melillo Giovanni
Published: August 2009

Introduction

Mechanisms of action of action of HIF‐1 inhibitors

Conclusions

Intratumour hypoxia has long been considered a driving force of tumour progression and a negative prognostic factor in human cancers. The discovery of hypoxia inducible factors (HIFs), which mediate transcriptional responses to changes in oxygen levels, has renewed enthusiasm for the discovery and development of targeted therapies exploiting the hypoxic tumour microenvironment. In spite of an ever increasing number of putative small molecule inhibitors of HIF, only few progress through pre‐clinical and early clinical development. In this review, we will focus primarily on: (1) HIF inhibitors that have been more recently described and (2) small molecules targeting HIF that are being tested in early clinical trials or that are already approved for use in patients. A rigorous ‘validation’ of HIF targeted therapies in relevant pre‐clinical models and eventually in pharmacodynamic‐based early clinical trials is essential for ‘credentialing’ HIF‐1 as a legitimate target that can be pharmacologically modulated in cancer patients.
The influence of a probiotic milk drink on the development of gingivitis: a pilot study
Authors: Staab Benedict , Eick Sigrun , Knöfler Gerhild , Jentsch Holger
Published: August 2009

Aim: The aim of this study was to determine the effect of a probiotic milk drink on gingival health and the development of experimental gingivitis.

Material and Methods: Fifty volunteer students took part in a parallel‐designed non‐blinded study. The test group drank a probiotic drink once a day; the control group did not receive any product to drink. After 8 weeks, individual mechanical plaque control was interrupted for 96 h. Papilla bleeding index, interproximal plaque and Turesky plaque index (PI) were recorded at baseline, after 8 weeks and again 96 h later. At the same time points, gingival crevicular fluid had been collected for analysis of polymorphonuclear elastase, myeloperoxidase (MPO) and matrix metalloproteinase‐3 (MMP‐3).

Results: Interproximal PI and papillary bleeding were not different between the groups. In the test group, elastase activity and MMP‐3 amount were significantly lower after the intake of the probiotic milk drink (p<0.001 and 0.016). There was a significant increase of MPO activity in the control group; both groups were different at the end of the study (p=0.014).

Conclusions: The data suggest a beneficial effect of the probiotic milk drink on gingival inflammation.
Postnatal handling reverses social anxiety in serotonin receptor 1A knockout mice
Authors: Zanettini C. , Carola V. , Lo Iacono L. , Moles A. , Gross C. , D'Amato F. R.
Published: August 2009

Mice lacking the serotonin receptor 1A (Htr1a knockout, Htr1aKO) show increased innate and conditioned anxiety. This phenotype depends on functional receptor activity during the third through fifth weeks of life and thus appears to be the result of long‐term changes in brain function as a consequence of an early deficit in serotonin signaling. To evaluate whether this phenotype can be influenced by early environmental factors, we subjected Htr1a knockout mice to postnatal handling, a procedure known to reduce anxiety‐like behavior and stress responses in adulthood. Offspring of heterozygous Htr1a knockout mice were separated from their mother and exposed 15 min each day from postnatal day 1 (PD1) to PD14 to clean bedding. Control animals were left undisturbed. Maternal behavior was observed during the first 13 days of life. Adult male offspring were tested in the open field, social approach and resident–intruder tests and assessed for corticosterone response to restraint stress. Knockout mice showed increased anxiety in the open field and in the social approach test as well as an enhanced corticosterone response to stress. However, while no effect of postnatal handling was seen in wild‐type mice, handling reduced anxiety‐like behavior in the social interaction test and the corticosterone response to stress in knockout mice. These findings extend the anxiety phenotype of Htr1aKO mice to include social anxiety and demonstrate that this phenotype can be moderated by early environmental factors.
The effect of mGlu8 deficiency in animal models of psychiatric diseases
Authors: Fendt M. , Bürki H. , Imobersteg S. , Van Der Putten H. , McAllister K. , Leslie J. C. , Shaw D. , Hölscher C.
Published: August 2009

The metabotropic glutamate receptor subtype 8 (mGlu8) is presynaptically located and regulates the release of the transmitter. Dysfunctions of this mechanism are involved in the pathophysiology of different psychiatric disorders. mGlu8 deficient mice have been previously investigated in a range of studies, but the results are contradictory and there are still many open questions. Therefore, we tested mGlu8‐deficient animals in different behavioral tasks that are commonly used in neuropsychiatric research. Our results show a robust contextual fear deficit in mGlu8‐deficient mice. Furthermore, novel object recognition, chlordiazepoxide‐facilitated extinction of operant conditioning and the acoustic startle response were attenuated by mGlu8 deficiency. We found no changes in sensory processing, locomotor activity, prepulse inhibition, phencyclidine‐induced changes in locomotion or prepulse inhibition, operant conditioning, conditioned fear to a discrete cue or in animal models of innate fear and post‐traumatic stress disorder. We conclude that mGlu8 might be a potential target for disorders with pathophysiological changes in brain areas where mGlu8 modulates glutamate and gamma‐amino butyric acid (GABA) transmission. Our data especially point to anxiety disorders involving exaggerated contextual fear, such as generalized anxiety disorders, and to conditions with disturbed declarative memory.
n‐3 Fatty acid supplementation in burned paediatric patients
Authors: Marín MC , Osimani NE , Rey GE , De Alaniz MJT
Published: August 2009

Aim:  To determine the effect of dietary supplementation with n‐3 fatty acids (FA) in paediatric burned patients who had less than 20% of total body surface affected.

Methods:  Burned patients were randomly assigned into two groups, one of them received a supplement of n‐3 FA during 5 weeks; the other group was considered as not n‐3 supplemented burned group. A third group of no burned patients was selected as control. Blood samples were collected at admission and in burned groups at the final of the study. Plasma and erythrocyte phospholipid FA composition and some biochemical parameters related to the clinical evolution: total plasma proteins and C3 and C4 complement proteins were determined.

Results:  In the early post‐burn patients, there is an increase in saturated and monounsaturated FAs in plasma phospholipids, and a decrease in polyunsaturated FAs compared with control. These alterations are in favour of proinflammatory response to burn injury. In n‐3 FA supplemented group, these changes were further reverted, and a favourable response in the amount of total plasma proteins and in C3 and C4 proteins of the complement system was demonstrated.

Conclusion:  Dietary n‐3 FA supplementation might be beneficial for patients suffering thermal injury.
Sharing the mind of Christ: preliminary thoughts on dementia and the Cross
Authors: Kevern Peter
Published: August 2009

The increasing prevalence of forms of dementia poses some profound challenges, not least to our Christology and soteriology. In particular, it exposes the degree to which faith, grace and salvation are all still linked to the concept of the self-conscious individual, and how this approach limits the range of possible theological responses to dementia. In this paper, the author argues that a theological response in depth requires us to consider the possibility of saying that Christ ‘demented’ on the Cross. Some implications of making such an assertion are explored, both for Christology in general and for the ways Christ may be spoken of as ‘present to’ those with dementia.
The carbohydrate‐binding module family 20 – diversity, structure, and function
Authors: Christiansen Camilla , Abou Hachem Maher , Janeček Štefan , Viksø‐Nielsen Anders , Blennow Andreas , Svensson Birte
Published: August 2009

Starch‐active enzymes often possess starch‐binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate‐binding module (CBM) families, and CBM20 is the earliest‐assigned and best characterized family. High diversity characterizes CBM20s, which occur in starch‐active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch‐phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily relationships. Data on binding to and enzymatic activity towards soluble ligands and starch granules are summarized for wild‐type, mutant and chimeric fusion proteins involving CBM20s. Noticeably, whereas CBM20s in amylolytic enzymes confer moderate binding affinities, with dissociation constants in the low micromolar range for the starch mimic β‐cyclodextrin, recent findings indicate that CBM20s in regulatory enzymes have weaker, low millimolar affinities, presumably facilitating dynamic regulation. Structures of CBM20s, including the first example of a full‐length glucoamylase featuring both the catalytic domain and the SBD, are summarized, and distinct architectural and functional features of the two SBDs and roles of pivotal amino acids in binding are described. Finally, some applications of SBDs as affinity or immobilization tags and, recently, in biofuel and in planta bioengineering are presented.
Capacity assessment under the Mental Capacity Act 2005: Delivering on the functional approach?
Authors: Donnelly Mary
Published: August 2009

A fundamental aspect of the Mental Capacity Act 2005 (MCA) is the statutory endorsement of a functional approach to capacity. In principle, this requires a separate assessment of capacity in respect of each decision to be made. Most capacity assessments take place at a non-judicial level, and, outside of day-to-day decisions, the most common assessors are likely to be healthcare professionals. This paper investigates the practical operation of the capacity assessment process at both judicial and non-judicial levels. It asks whether the process can deliver on the MCA's goal of preserving maximum decision-making freedom, while, at the same time, providing an appropriate degree of protection. It argues that assessors who are not legally trained encounter significant difficulties in carrying out the essentially legal task of assessing capacity. It also shows that assessors' values and biases, both professional and personal, are likely to impact on the conclusions reached. Having identified these difficulties, the paper considers their implications for the MCA's approach to capacity. It evaluates the attempts in the MCA itself to address assessors' limitations and argues that these will not have a substantial impact on the way in which capacity assessors operate. It considers other ways in which the quality of assessment could be improved and puts forward specific suggestions as to how greater assessor accountability can be delivered and increased rigour introduced to the capacity assessment process.
Debt investment as a tool for value transfer in biodiversity conservation
Authors: Mandel James T. , Donlan C. Josh , Wilcox Chris , Cudney‐Bueno Richard , Pascoe Sean , Tulchin Drew
Published: August 2009

A central challenge in conservation is to create value effectively around local resources that will lead to better environmental stewardship. Historically, conservationists have either used indirect approaches, such as the promotion of alternate industries like eco‐tourism, or more recently direct approaches, such as land purchases and cash payments. While direct payment programs, through conservation incentive agreements and regular payments for ecosystem preservation, are being trialed in low‐income nations, the lack of enforceable property rights and contractual laws can present challenges when trying to influence conservation outcomes in local communities. We suggest an alternative approach—debt‐based investment—that capitalizes environmental assets locally and makes that capital available to local communities through collateralized lending, microfinance approaches, and access to affordable financial services. Tying the value of capital in a conservation lending trust to the global value for intact environmental resources will create incentives for local environmental stewardship while providing economic access to what is often a poor community's most valuable asset—intact natural resources.
Response of methanogenic archaeal community to nitrate addition in rice field soil
Authors: Yuan Quan , Lu Yahai
Published: August 2009

Addition of nitrate strongly inhibits CH4 production from anoxic soil. The main mechanisms were considered to be the substrate competition and the toxic effect of denitrification intermediates on the methanogenesis. However, it is unclear whether these inhibitory effects are reflected in the structure and dynamics of methanogenic community in the soil. In the present study, the response of methanogenic archaeal community to nitrate addition was determined using terminal restriction fragment length polymorphisms in combination with cloning and sequencing of archaeal 16S rRNA genes. When nitrate was added at the beginning of an anoxic incubation of rice field soil, denitrification occurred rapidly and the denitrification intermediates were detected only for a short time. Total production of CH4 was reduced, but no obvious effect on the structure of methanogenic community was observed. In contrast, when nitrate was added 20 days after the anoxic incubation, the denitrification intermediates obviously accumulated. CH4 production was completely suppressed for 7 and 16 days from treatments of 5 and 10 mM nitrate respectively. The dynamics of methanogenic community also diverged greatly from the control. While the hydrogenotrophic methanogens increased and the acetoclastic methanogens decreased with the incubation in the control soil, the structure and abundance of the methanogenic community remained unchanged after the addition of nitrate. Methanogenesis resumed when the denitrification intermediates were depleted in soil. The analysis of carbon isotopic signals revealed that hydrogenotrophic methanogenesis recovered faster than acetoclastic methanogenesis. Our study suggests that the accumulation of denitrification intermediates has a strong inhibitory effect on the activity but not the structure of methanogenic community.
Evidence for a release of brain‐derived neurotrophic factor from the brain during exercise
Authors: Rasmussen Peter , Brassard Patrice , Adser Helle , Pedersen Martin V. , Leick Lotte , Hart Emma , Secher Niels H. , Pedersen Bente K. , Pilegaard Henriette
Published: August 2009

Brain‐derived neurotrophic factor (BDNF) has an important role in regulating maintenance, growth and survival of neurons. However, the main source of circulating BDNF in response to exercise is unknown. To identify whether the brain is a source of BDNF during exercise, eight volunteers rowed for 4 h while simultaneous blood samples were obtained from the radial artery and the internal jugular vein. To further identify putative cerebral region(s) responsible for BDNF release, mouse brains were dissected and analysed for BDNF mRNA expression following treadmill exercise. In humans, a BDNF release from the brain was observed at rest (P < 0.05), and increased two‐ to threefold during exercise (P < 0.05). Both at rest and during exercise, the brain contributed 70–80% of circulating BDNF, while that contribution decreased following 1 h of recovery. In mice, exercise induced a three‐ to fivefold increase in BDNF mRNA expression in the hippocampus and cortex, peaking 2 h after the termination of exercise. These results suggest that the brain is a major but not the sole contributor to circulating BDNF. Moreover, the importance of the cortex and hippocampus as a source for plasma BDNF becomes even more prominent in response to exercise.
Is there an interaction between PPARD T294C and PPARGC1A Gly482Ser polymorphisms and human endurance performance?
Authors: Eynon Nir , Meckel Yoav , Alves Alberto Jorge , Yamin Chen , Sagiv Michael , Goldhammer Ehud , Sagiv Moran
Published: August 2009

Functional Gly482Ser (rs8192678) and T294C (rs2016520) polymorphisms in the peroxisome proliferator‐activated receptor γ coactivator‐1 (PPARGC1A) and peroxisome proliferator‐activated receptor δ (PPARD) genes, respectively, have been associated with mRNA and/or protein activity. The aim of this study was to determine their frequency distribution among 155 Israeli athletes (endurance athletes and sprinters) and 240 healthy control subjects. There were no differences between the endurance athletes, the sprinters and the control group across the PPARD T294C genotypes (P= 0.62). Similarly, no statistical differences were found between the subgroups of elite‐level endurance athletes (those who had represented Israel in a world track and field championship or in the Olympic Games) and national‐level endurance athletes (P= 0.3), or between elite‐level and national‐level sprinters (P= 0.9). However, a combined influence of these two polymorphisms on endurance performance was found. The PPARD CC +PPARGC1A Gly/Gly genotypes were more frequently found in the elite endurance athletes than in national‐level endurance athletes (P < 0.000). In the cohort of endurance athletes, the odds ratio of the ‘optimal genotype’ for endurance athletes (PPARD CC +PPARGC1A Gly/Gly +PPARGC1A Gly/Ser) being an elite‐level athlete was 8.32 (95% confidence interval 2.2–31.4). In conclusion, the present study suggests that PPARD T294C is not associated with endurance performance. However, a higher frequency of the PPARGC1A Gly/Gly +PPARD CC genotype is associated with elite‐level endurance athletes.
Soap Operas to Save Energy
Authors: Charles Dan
Published: August 2009

Filmmaker John Johnson is collaborating with the creators of popular video programs on the Web to develop scripts that show people conserving energy and water and considering how their consumption choices might affect the planet.
Conjugative Plasmid fromLactobacillus gasseriLA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A
Authors: Ito Yoshiyuki , Kawai Yasushi , Arakawa Kensuke , Honme Yoshiko , Sasaki Takashi , Saito Tadao
Published: August 2009

Gassericin A is a circular bacteriocin produced byLactobacillus gasseristrain LA39. We found a 33,333-bp plasmid, designated pLgLA39, in this strain. pLgLA39 contained 44 open reading frames, including seven genes related to gassericin A production/immunity (gaa), as well as genes for replication, plasmid maintenance, and conjugative transfer. pLgLA39 was transferred from LA39 to the type strain ofL. gasseri(JCM 1131) by filter mating. The transconjugant exhibited >30-fold-higher more resistance to gassericin A and produced antibacterial activity.Lactobacillus reuteriLA6, the producer of reutericin 6, was proved to harbor a plasmid indistinguishable from pLgLA39 and carrying seven genes 100% identical togaa. This suggests that pLgLA39 might have been transferred naturally betweenL. gasseriLA39 andL. reuteriLA6. The sevengaagenes of pLgLA39 were cloned into a plasmid vector to construct pGAA. JCM 1131Ttransformed with pGAA expressed antibacterial activity and resistance to gassericin A. pGAA was segregationally more stable than a pGAA derivative plasmid from whichgaaAwas deleted and even was more stable than the vector. This suggests the occurrence of postsegregational host killing by thegaagenes. pLgLA39 carried apemIKhomolog, and segregational stabilization of a plasmid by the pLgLA39-typepemIKgenes was also confirmed. Thus, pLgLA39 was proved to carry the genes for at least two plasmid maintenance mechanisms, i.e.,gaaandpemIK. Plasmids containing arepAgene similar to pLgLA39repAwere distributed in severalL. gasseristrains.
Transcriptional and Functional Studies of
Acidithiobacillus ferrooxidans
Genes Related to Survival in the Presence of Copper
Authors: Navarro Claudio A. , Orellana Luis H. , Mauriaca Cecilia , Jerez Carlos A.
Published: August 2009

The acidophilic
Acidithiobacillus ferrooxidans
can resist exceptionally high copper (Cu) concentrations. This property is important for its use in biomining processes, where Cu and other metal levels range usually between 15 and 100 mM. To learn about the mechanisms that allow
A. ferrooxidans
cells to survive in this environment, a bioinformatic search of its genome showed the presence of at least 10 genes that are possibly related to Cu homeostasis. Among them are three genes coding for putative ATPases related to the transport of Cu (
A. ferrooxidans copA1
[

copA1
Af

],

copA2
Af

, and

copB
Af

), three genes related to a system of the resistance nodulation cell division family involved in the extraction of Cu from the cell (

cusA
Af

,

cusB
Af

, and

cusC
Af

), and two genes coding for periplasmic chaperones for this metal (

cusF
Af

and

copC
Af

). The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR using
A. ferrooxidans
cells adapted for growth in the presence of high concentrations of Cu. The putative
A. ferrooxidans
Cu resistance determinants were found to be upregulated when this bacterium was exposed to Cu in the range of 5 to 25 mM. These
A. ferrooxidans
genes conferred to
Escherichia coli
a greater Cu resistance than wild-type cells, supporting their functionality. The results reported here and previously published data strongly suggest that the high resistance of the extremophilic
A. ferrooxidans
to Cu may be due to part or all of the following key elements: (i) a wide repertoire of Cu resistance determinants, (ii) the duplication of some of these Cu resistance determinants, (iii) the existence of novel Cu chaperones, and (iv) a polyP-based Cu resistance system.
Intra- and Interspecies Conjugal Transfer of Tn
916
-Like Elements from
Lactococcus lactis
In Vitro and In Vivo
Authors: Boguslawska Joanna , Zycka-Krzesinska Joanna , Wilcks Andrea , Bardowski Jacek
Published: August 2009

Tetracycline-resistant
Lactococcus lactis
strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed
L. lactis
isolates were able to transfer tetracycline resistance determinants in vitro to
L. lactis
Bu2-60, at frequencies ranging from 10
−5
to 10
−7
transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to
Enterococcus faecalis
JH2-2, whereas no transfer to
Bacillus subtilis
YBE01,
Pseudomonas putida
KT2442,
Agrobacterium tumefaciens
UBAPF2, or
Escherichia coli
JE2571 was observed. Rats were initially inoculated with the recipient
E. faecalis
strain JH2-2, and after a week, the
L. lactis
IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the
tet
(M) gene showed that this gene was identical to
tet
(M) localized on the conjugative transposon Tn
916
. Primer-specific PCR confirmed that the Tn
916
transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn
916
-like antibiotic resistance transposon from
L. lactis
to
E. faecalis
. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria.
Community Structure and Dynamics of Small Eukaryotes Targeted by New Oligonucleotide Probes: New Insight into the Lacustrine Microbial Food Web
Authors: Mangot Jean-François , Lepère Cécile , Bouvier Christophe , Debroas Didier , Domaizon Isabelle
Published: August 2009

The seasonal dynamics of the small eukaryotic fraction (cell diameter, 0.2 to 5 μm) was investigated in a mesotrophic lake by tyramide signal amplification-fluorescence in situ hybridization targeting seven different phylogenetic groups: Chlorophyceae, Chrysophyceae, Cryptophyceae, Cercozoa, LKM11, Perkinsozoa (two clades), and Fungi. The abundance of small eukaryotes ranged from 1,692 to 10,782 cells ml
−1
. The dominant groups were the Chrysophyceae and the Chlorophyceae, which represented 19.6% and 17.9% of small eukaryotes, respectively. The results also confirmed the quantitative importance of putative parasites, Fungi and Perkinsozoa, in the small heterotrophic eukaryotic assemblage. The relative abundances recorded for the Perkinsozoa group reached as much as 31.6% of total targeted eukaryotes during the summer. The dynamics of Perkinsozoa clade 1 coincided with abundance variations in
Peridinium
and
Ceratium
spp. (Dinoflagellates), while the dynamics of Perkinsozoa clade 2 was linked to the presence of
Dinobryon
spp. (Chrysophyceae). Fungi, represented by chytrids, reached maximal abundance in December (569 cells ml
−1
) and were mainly correlated with the dynamics of diatoms, especially
Melosira varians
. A further new finding of this study is the recurrent presence of Cercozoa (6.2%) and LKM11 (4.5%) cells. This quantitative approach based on newly designed probes offers a promising means of in-depth analysis of microbial food webs in lakes, especially by revealing the phylogenetic composition of the small heterotrophic flagellate assemblage, for which an important fraction of cells are generally unidentified by classical microscopy (on average, 96.8% of the small heterotrophic flagellates were identified by the specific probes we used in this study).
Evolved β-Galactosidases from
Geobacillus stearothermophilus
with Improved Transgalactosylation Yield for Galacto-Oligosaccharide Production
Authors: Placier Gaël , Watzlawick Hildegard , Rabiller Claude , Mattes Ralf
Published: August 2009

A mutagenesis approach was applied to the β-galactosidase BgaB from
Geobacillus stearothermophilus
KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined. An change of one arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved β-galactosidases, a major trisaccharide was formed. Its structure was characterized as β-
d
-galactopyranosyl-(1→3)-β-
d
-galactopyranosyl-(1→4)-
d
-glucopyranoside (3′-galactosyl-lactose). At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3′-galactosyl-lactose production of 23%.
New Plasmid Tools for Genetic Analysis of
Actinobacillus pleuropneumoniae
and Other
Pasteurellaceae
Authors: Bossé Janine T. , Durham Andrew L. , Rycroft Andrew N. , Kroll J. Simon , Langford Paul R.
Published: August 2009

We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of
Actinobacillus pleuropneumoniae
and other members of the
Pasteurellaceae
. A tandem reporter plasmid, pMC-Tandem, carrying promoterless
xylE
and
gfpmut3
genes downstream of a multiple-cloning site (MCS), can be used for identification of transcriptional regulators and conditions which favor gene expression from different cloned promoters. The ability to detect transcriptional regulators using the tandem reporter system was validated in
A. pleuropneumoniae
using the cloned
rpoE
(σ
E
) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of σ
E
, among a bank of random transposon mutants, as well as to detect induction of σ
E
following exposure of
A. pleuropneumoniae
to ethanol or heat shock. pMCsodCP, carrying the cloned
sodC
promoter of
A. pleuropneumoniae
, was functional in
A. pleuropneumoniae
,
Haemophilus influenzae
,
Haemophilus parasuis
,
Mannheimia haemolytica
, and
Pasteurella multocida
. Two general expression vectors, pMK-Express and pMC-Express, which differ in their antibiotic resistance markers (kanamycin and chloramphenicol, respectively), were constructed for the
Pasteurellaceae
. Both plasmids have the
A. pleuropneumoniae sodC
promoter upstream of the
gfpmut3
gene and an extended MCS. Replacement of
gfpmut3
with a gene of interest allows complementation and heterologous gene expression, as evidenced by expression of the
Haemophilus ducreyi nadV
gene in
A. pleuropneumoniae
, rendering the latter NAD independent.
Inorganic Phosphate and Sodium Ions Are Cogerminants for Spores of
Clostridium perfringens
Type A Food Poisoning-Related Isolates
Authors: Paredes-Sabja Daniel , Udompijitkul Pathima , Sarker Mahfuzur R.
Published: August 2009

Clostridium perfringens
type A isolates carrying a chromosomal copy of the enterotoxin (
cpe
) gene are involved in the majority of food poisoning (FP) outbreaks, while type A isolates carrying a plasmid-borne
cpe
gene are involved in
C. perfringens
-associated non-food-borne (NFB) gastrointestinal diseases. To cause diseases,
C. perfringens
spores must germinate and return to active growth. Previously, we showed that only spores of FP isolates were able to germinate with K
+
ions. We now found that the spores of the majority of FP isolates, but none of the NFB isolates, germinated with the cogerminants Na
+
and inorganic phosphate (NaP
i
) at a pH of ∼6.0. Spores of
gerKA-KC
and
gerAA
mutants germinated to a lesser extent and released less dipicolinic acid (DPA) than did wild-type spores with NaP
i
. Although
gerKB
spores germinated to a similar extent as wild-type spores with NaP
i
, their rate of germination was lower. Similarly,
gerO
and
gerO gerQ
mutant spores germinated slower and released less DPA than did wild-type spores with NaP
i
. In contrast,
gerQ
spores germinated to a slightly lesser extent than wild-type spores but released all of their DPA during NaP
i
germination. In sum, this study identified NaP
i
as a novel nutrient germinant for spores of most FP isolates and provided evidence that proteins encoded by the
gerKA-KC
operon,
gerAA
, and
gerO
are required for NaP
i
-induced spore germination.
Transcriptomic Response of
Escherichia coli
O157:H7 to Oxidative Stress
Authors: Wang Siyun , Deng Kaiping , Zaremba Sam , Deng Xiangyu , Lin Chiahui , Wang Qian , Tortorello Mary Lou , Zhang Wei
Published: August 2009

Chlorinated water is commonly used in industrial operations to wash and sanitize fresh-cut, minimally processed produce. Here we compared 42 human outbreak strains that represented nine distinct
Escherichia coli
O157:H7 genetic lineages (or clades) for their relative resistance to chlorine treatment. A quantitative measurement of resistance was made by comparing the extension of the lag phase during growth of each strain under exposure to sublethal concentrations of sodium hypochlorite in Luria-Bertani or brain heart infusion broth. Strains in clade 8 showed significantly (
P
< 0.05) higher resistance to chlorine than strains from other clades of
E. coli
O157:H7. To further explore how
E. coli
O157:H7 responds to oxidative stress at transcriptional levels, we analyzed the global gene expression profiles of two strains, TW14359 (clade 8; associated with the 2006 spinach outbreak) and Sakai (clade 1; associated with the 1996 radish sprout outbreak), under sodium hypochlorite or hydrogen peroxide treatment. We found over 380 genes were differentially expressed (more than twofold;
P
< 0.05) after exposure to low levels of chlorine or hydrogen peroxide. Significantly upregulated genes included several regulatory genes responsive to oxidative stress, genes encoding putative oxidoreductases, and genes associated with cysteine biosynthesis, iron-sulfur cluster assembly, and antibiotic resistance. Identification of
E. coli
O157:H7 strains with enhanced resistance to chlorine decontamination and analysis of their transcriptomic response to oxidative stress may improve our basic understanding of the survival strategy of this human enteric pathogen on fresh produce during minimal processing.
Quantification of Horizontal Transmission of
Salmonella enterica
Serovar Enteritidis Bacteria in Pair-Housed Groups of Laying Hens
Authors: Thomas M. E. , Klinkenberg D. , Ejeta G. , Van Knapen F. , Bergwerff A. A. , Stegeman J. A. , Bouma A.
Published: August 2009

An important source of human salmonellosis is the consumption of table eggs contaminated with
Salmonella enterica
serovar Enteritidis. Optimization of the various surveillance programs currently implemented to reduce human exposure requires knowledge of the dynamics of
S.
Enteritidis infection within flocks. The aim of this study was to provide parameter estimates for a transmission model of
S.
Enteritidis in laying-type chicken flocks. An experiment was carried out with 60 pairs of laying hens. Per pair, one hen was inoculated with
S.
Enteritidis and the other was contact exposed. After inoculation, cloacal swab samples from all hens were collected over 18 days and tested for the presence of
S.
Enteritidis. On the basis of this test, it was determined if and when each contact-exposed hen became colonized. A transmission model including a latency period of 1 day and a slowly declining infectivity level was fitted. The mean initial transmission rate was estimated to be 0.47 (95% confidence interval [CI], 0.30 to 0.72) per day. The reproduction number
R
0
, the average number of hens infected by one colonized hen in a susceptible population, was estimated to be 2.8 (95% CI, 1.9 to 4.2). The generation time, the average time between colonization of a “primary” hen and colonization of contact-exposed hens, was estimated to be 7.0 days (95% CI, 5.0 to 11.6 days). Simulations using these parameters showed that a flock of 20,000 hens would reach a maximum colonization level of 92% within 80 days after colonization of the first hen. These results can be used, for example, to evaluate the effectiveness of control and surveillance programs and to optimize these programs in a cost-benefit analysis.
Impaired Uptake and/or Utilization of Leucine bySaccharomyces cerevisiaeIs Suppressed by theSPT15-300Allele of the TATA-Binding Protein Gene
Authors: Baerends Richard J. S. , Qiu Jin-Long , Rasmussen Simon , Nielsen Henrik Bjørn , Brandt Anders
Published: August 2009

Successful fermentations to produce ethanol require microbial strains that have a high tolerance to glucose and ethanol. Enhanced glucose/ethanol tolerance of the laboratory yeastSaccharomyces cerevisiaestrain BY4741 under certain growth conditions as a consequence of the expression of a dominant mutant allele of theSPT15gene (SPT15-300) corresponding to the three amino acid changes F177S, Y195H, and K218R has been reported (H. Alper, J. Moxley, E. Nevoigt, G. R. Fink, and G. Stephanopoulos, Science 314:1565-1568, 2006). TheSPT15gene codes for the TATA-binding protein. This finding prompted us to examine the effect of expression of theSPT15-300allele in various yeast species of industrial importance. Expression ofSPT15-300in leucine-prototrophic strains ofS. cerevisiae,Saccharomyces bayanus, orSaccharomyces pastorianus(lager brewing yeast), however, did not improve tolerance to ethanol on complex rich medium (yeast extract-peptone-dextrose). The enhanced growth of the laboratory yeast strain BY4741 expressing theSPT15-300mutant allele was seen only on defined media with low concentrations of leucine, indicating that the apparent improved growth in the presence of ethanol was indeed associated with enhanced uptake and/or utilization of leucine. Reexamination of the microarray data published by Alper and coworkers likewise suggested that expression of genes coding for the leucine permeases, Tat1p and Bap3p, were upregulated in theSPT15-300mutant, as was expression of the genesARO10,ADH3,ADH5, andSFA1, involved in leucine degradation.
Poly(3-Hydroxybutyrate) Production from Glycerol by
Zobellella denitrificans
MW1 via High-Cell-Density Fed-Batch Fermentation and Simplified Solvent Extraction
Authors: Ibrahim Mohammad H. A. , Steinbüchel Alexander
Published: August 2009

Industrial production of biodegradable polyesters such as polyhydroxyalkanoates is hampered by high production costs, among which the costs for substrates and for downstream processing represent the main obstacles. Inexpensive fermentable raw materials such as crude glycerol, an abundant by-product of the biodiesel industry, have emerged to be promising carbon sources for industrial fermentations. In this study,
Zobellella denitrificans
MW1, a recently isolated bacterium, was used for the production of poly(3-hydroxybutyrate) (PHB) from glycerol as the sole carbon source. Pilot-scale fermentations (42-liter scale) were conducted to scale up the high PHB accumulation capability of this strain. By fed-batch cultivation, at first a relatively high cell density (29.9 ± 1.3 g/liter) was obtained during only a short fermentation period (24 h). However, the PHB content was relatively low (31.0% ± 4.2% [wt/wt]). Afterwards, much higher concentrations of PHB (up to 54.3 ± 7.9 g/liter) and higher cell densities (up to 81.2 ± 2.5 g/liter) were obtained by further fed-batch optimization in the presence of 20 g/liter NaCl, with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. A high specific growth rate (0.422/h) and a short doubling time (1.64 h) were attained. The maximum PHB content obtained was 66.9% ± 7.6% of cell dry weight, and the maximum polymer productivity and substrate yield coefficient were 1.09 ± 0.16 g/liter/h and 0.25 ± 0.04 g PHB/g glycerol, respectively. Furthermore, a simple organic solvent extraction process was employed for PHB recovery during downstream processing: self-flotation of cell debris after extraction of PHB with chloroform allowed a convenient separation of a clear PHB-solvent solution from the cells. Maximum PHB recovery (85.0% ± 0.10% [wt/wt]) was reached after 72 h of extraction with chloroform at 30°C, with a polymer purity of 98.3% ± 1.3%.
Surface Colonization by Marine Roseobacters: Integrating Genotype and Phenotype
Authors: Slightom Rachael N. , Buchan Alison
Published: August 2009

TheRoseobacterclade is a broadly distributed, abundant, and biogeochemically relevant group of marine bacteria. Representatives are often associated with organic surfaces in disparate marine environments, suggesting that a sessile lifestyle is central to the ecology of lineage members. The importance of surface association and colonization has been demonstrated recently for select strains, and it has been hypothesized that production of antimicrobial agents, cell density-dependent regulatory mechanisms, and morphological features contribute to the colonization success of roseobacters. Drawing on these studies, insight into a broad representation of strains is facilitated by the availability of a substantial collection of genome sequences that provides a holistic view of these features among clade members. These genome data often corroborate phenotypic data but also reveal significant variation in terms of gene content and synteny among group members, even among closely related strains (congeners and conspecifics). Thus, while detailed studies of representative strains are serving as models for how roseobacters transition between planktonic and sessile lifestyles, it is becoming clear that additional studies are needed if we are to have a more comprehensive view of how these transitions occur in different lineage members. This is important if we are to understand how associations with surfaces influence metabolic activities contributing to the cycling of carbon and nutrients in the world's oceans.
Actions of
Mycobacterium
sp. Strain AP1 on the Saturated- and Aromatic-Hydrocarbon Fractions of Fuel Oil in a Marine Medium
Authors: Vila Joaquim , Grifoll Magdalena
Published: August 2009

The pyrene-degrading
Mycobacterium
sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C
12
to C
40
) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(
a
)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by
Mycobacterium
sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.
Candida albicans
Hyphal Formation and Virulence Assessed Using a
Caenorhabditis elegans
Infection Model
Authors: Pukkila-Worley Read , Peleg Anton Y. , Tampakakis Emmanouil , Mylonakis Eleftherios
Published: August 2009

Candida albicans
colonizes the human gastrointestinal tract and can cause life-threatening systemic infection in susceptible hosts. We study here
C. albicans
virulence determinants using the nematode
Caenorhabditis elegans
in a pathogenesis system that models candidiasis. The yeast form of
C. albicans
is ingested into the
C. elegans
digestive tract. In liquid media, the yeast cells then undergo morphological change to form hyphae, which results in aggressive tissue destruction and death of the nematode. Several lines of evidence demonstrate that hyphal formation is critical for
C. albicans
pathogenesis in
C. elegans
. First, two yeast species unable to form hyphae (
Debaryomyces hansenii
and
Candida lusitaniae
) were less virulent than
C. albicans
in the
C. elegans
assay. Second, three
C. albicans
mutant strains compromised in their ability to form hyphae (
efg1Δ/efg1Δ
,
flo8Δ/flo8Δ
, and
cph1Δ/cph1Δ efg1Δ/efg1Δ
) were dramatically attenuated for virulence. Third, the conditional
tet-NRG1
strain, which enables the external manipulation of morphogenesis in vivo, was more virulent toward
C. elegans
when the assay was conducted under conditions that permit hyphal growth. Finally, we demonstrate the utility of the
C. elegans
assay in a screen for
C. albicans
virulence determinants, which identified several genes important for both hyphal formation in vivo and the killing of
C. elegans
, including the recently described
CAS5
and
ADA2
genes. These studies in a
C. elegans-C. albicans
infection model provide insights into the virulence mechanisms of an important human pathogen.
Trypanosomatid Genomes Contain Several Subfamilies of ingi-Related Retroposons
Authors: Bringaud Frédéric , Berriman Matthew , Hertz-Fowler Christiane
Published: August 2009

Retroposons are ubiquitous transposable elements found in the genomes of most eukaryotes, including trypanosomatids. The African and American trypanosomes (
Trypanosoma brucei
and
Trypanosoma cruzi
) contain long autonomous retroposons of the ingi clade (Tbingi and L1Tc, respectively) and short nonautonomous truncated versions (TbRIME and NARTc, respectively), as well as degenerate ingi-related retroposons devoid of coding capacity (DIREs). In contrast,
Leishmania major
contains only remnants of extinct retroposons (LmDIREs) and of short nonautonomous heterogeneous elements (LmSIDERs). We extend this comparative and evolutionary analysis of retroposons to the genomes of two other African trypanosomes (
Trypanosoma congolense
and
Trypanosoma vivax
) and another
Leishmania
sp. (
Leishmania braziliensis
). Three new potentially functional retroposons of the ingi clade have been identified: Tvingi in
T. vivax
and Tcoingi and L1Tco in
T. congolense. T. congolense
is the first trypanosomatid containing two classes of potentially active retroposons of the ingi clade. We analyzed sequences located upstream of these new long autonomous ingi-related elements, which code for the recognition site of the retroposon-encoded endonuclease. The closely related Tcoingi and Tvingi elements show the same conserved pattern, indicating that the Tcoingi- and Tvingi-encoded endonucleases share site specificity. Similarly, the conserved pattern previously identified upstream of L1Tc has also been detected at the same relative position upstream of L1Tco elements. A phylogenetic analysis of all ingi-related retroposons identified so far, including DIREs, clearly shows that several distinct subfamilies have emerged and coexisted, though in the course of trypanosomatid evolution, only a few have been maintained as active elements in modern trypanosomatid (sub)species.