Genetic basis of photosynthetic responses to cold in two locally adapted populations of Arabidopsis thaliana.

Local adaptation is common, but the traits and genes involved are often unknown. Physiological responses to cold probably contribute to local adaptation in wide-ranging species, but the genetic basis underlying natural variation in these traits has rarely been studied. Using a recombinant inbred (495 lines) mapping population from locally adapted populations of Arabidopsis thaliana from Sweden and Italy, we grew plants at low temperature and mapped quantitative trait loci (QTLs) for traits related to photosynthesis: maximal quantum efficiency (Fv/Fm), rapidly reversible photoprotection (NPQfast), and photoinhibition of PSII (NPQslow) using high-throughput, whole-plant measures of chlorophyll fluorescence. In response to cold, the Swedish line had greater values for all traits, and for every trait, large effect QTLs contributed to parental differences. We found one major QTL affecting all traits, as well as unique major QTLs for each trait. Six trait QTLs overlapped with previously published locally adaptive QTLs based on fitness measured in the native environments over 3 years. Our results demonstrate that photosynthetic responses to cold can vary dramatically within a species, and may predominantly be caused by a few QTLs of large effect. Some photosynthesis traits and QTLs probably contribute to local adaptation in this system.

Genomic diversity and differentiation of a managed island wild boar population.

The evolution of island populations in natural systems is driven by local adaptation and genetic drift. However, evolutionary pathways may be altered by humans in several ways. The wild boar (WB) (Sus scrofa) is an iconic game species occurring in several islands, where it has been strongly managed since prehistoric times. We examined genomic diversity at 49 803 single-nucleotide polymorphisms in 99 Sardinian WBs and compared them with 196 wild specimens from mainland Europe and 105 domestic pigs (DP; 11 breeds). High levels of genetic variation were observed in Sardinia (80.9% of the total number of polymorphisms), which can be only in part associated to recent genetic introgression. Both Principal Component Analysis and Bayesian clustering approach revealed that the Sardinian WB population is highly differentiated from the other European populations (FST=0.126-0.138), and from DP (FST=0.169). Such evidences were mostly unaffected by an uneven sample size, although clustering results in reference populations changed when the number of individuals was standardized. Runs of homozygosity (ROHs) pattern and distribution in Sardinian WB are consistent with a past expansion following a bottleneck (small ROHs) and recent population substructuring (highly homozygous individuals). The observed effect of a non-random selection of Sardinian individuals on diversity, FST and ROH estimates, stressed the importance of sampling design in the study of structured or introgressed populations. Our results support the heterogeneity and distinctiveness of the Sardinian population and prompt further investigations on its origins and conservation status.

DAT isn't all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling.

The initial reinforcing properties of drugs of abuse, such as cocaine, are largely attributed to their ability to activate the mesolimbic dopamine system. Resulting increases in extracellular dopamine in the nucleus accumbens (NAc) are traditionally thought to result from cocaine's ability to block dopamine transporters (DATs). Here we demonstrate that cocaine also interacts with the immunosurveillance receptor complex, Toll-like receptor 4 (TLR4), on microglial cells to initiate central innate immune signaling. Disruption of cocaine signaling at TLR4 suppresses cocaine-induced extracellular dopamine in the NAc, as well as cocaine conditioned place preference and cocaine self-administration. These results provide a novel understanding of the neurobiological mechanisms underlying cocaine reward/reinforcement that includes a critical role for central immune signaling, and offer a new target for medication development for cocaine abuse treatment.

Human GII.4 norovirus VLP induces membrane invaginations on giant unilamellar vesicles containing secretor gene dependent α1,2-fucosylated glycosphingolipids.

Norovirus is a non-enveloped virus causing acute gastroenteritis. For human norovirus, no simple cell culture system is available and consequently knowledge on cellular entry of the virus is limited. The virus binds to ABH histo-blood group glycans on glycoproteins and glycosphingolipids. Non-secretors, characterized by the lack of ABH histo-blood group glycans in the gastrointestinal tract, are resistant to most norovirus infections, suggesting that these glycans may be part of the viral receptor. Recent studies have shown that polyomavirus enters the cell via membrane invaginations induced by the multivalent binding of the virus to receptor glycosphingolipids. In this study, we have investigated whether norovirus has the ability to induce membrane invaginations on giant unilamellar vesicles (GUVs) containing purified glycosphingolipids. First, we characterized the glycosphingolipid binding pattern of VLPs from the Dijon strain (genogroup II.4), using thin-layer chromatography. The VLP recognized the ABH active glycosphingolipids H type 1, Lewis b, B type 1, A type 1 and A Lewis b, but not lactotetraosylceramide or Lewis a, typically found in non-secretors. The binding pattern to glycosphingolipids incorporated into GUVs was in full agreement with the thin-layer chromatography experiments. Upon binding to the vesicles, the VLPs formed highly mobile clusters on the surface of the GUVs. VLP containing tubular invaginations were seen on the GUVs containing glycosphingolipids recognized by the VLP. In conclusion, this study suggests that human norovirus has the ability to induce membrane curvature by binding to and clustering glycosphingolipids, which may reflect the first step in cellular entry of the virus.

Interaction of virions with membrane glycolipids.

Cellular membranes contain various lipids including glycolipids (GLs). The hydrophilic head groups of GLs extend from the membrane into the aqueous environment outside the cell where they act as recognition sites for specific interactions. The first steps of interaction of virions with cells often include contacts with GLs. To clarify the details of such contacts, we have used the total internal reflection fluorescence microscopy to explore the interaction of individual unlabelled virus-like particles (or, more specifically, norovirus protein capsids), which are firmly bound to a lipid bilayer, and fluorescent vesicles containing glycosphingolipids (these lipids form a subclass of GLs). The corresponding binding kinetics were earlier found to be kinetically limited, while the detachment kinetics were logarithmic over a wide range of time. Here, the detachment rate is observed to dramatically decrease with increasing concentration of glycosphingolipids from 1% to 8%. This effect has been analytically explained by using a generic model describing the statistics of bonds in the contact area between a virion and a lipid membrane. Among other factors, the model takes the formation of GL domains into account. Our analysis indicates that in the system under consideration, such domains, if present, have a characteristic size smaller than the contact area between the vesicle and the virus-like particle.

Interaction of single viruslike particles with vesicles containing glycosphingolipids.

Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.

Major ampullate spidroins from Euprosthenops australis: multiplicity at protein, mRNA and gene levels.

Spider dragline silk possesses extraordinary mechanical properties. It consists of large fibrous proteins called spidroins that display modular structures. It is known to consist of two proteins: the major ampullate spidroin (MaSp) 1 and MaSp2. This study analyses MaSp sequences from the nursery-web spider Euprosthenops australis. We have identified a previously uncharacterized MaSp2 sequence and a new MaSp-like spidroin, which display distinct homogenous submotifs within their respective Gly-rich repeats. Furthermore, a group of MaSp1 cDNA clones show unexpected heterogeneity. Genomic PCR identified several MaSp1 gene variants within individual spiders, which suggests the presence of a gene cluster in E. australis. Finally, the evolution of spidroin genes is discussed in relation to phylogenetic analysis of nonrepetitive C-terminal domains from diverse species.

Training surgeons in endoscopic retrograde cholangiopancreatography.

BACKGROUND: General surgeons commonly perform upper gastrointestinal endoscopy in practice, but few perform endoscopic retrograde cholangiopancreatography (ERCP), partly because of limited training opportunities. This report focuses on the value of an ERCP fellowship training program to a broad-based, mature residency in surgery and our observations on the experience required for surgeons to be trained in advanced interventional ERCP. METHODS: Since the program was initiated in 1992, 13 ERCP fellows have been trained for individual periods of 6 to 14 months. This study investigated all procedures with fellow involvement (2,008 cases) from among a total experience of 3,641 ERCPs. Data collected included type of ERCP (diagnostic/therapeutic), fellow success in cannulating the duct of interest, and faculty success in cases of fellows who failed. Of the 13 fellows, 9 had previous endoscopy experience, but none had training in ERCP. RESULTS: An 85% cannulation rate was accepted as successful, and cannulation rates for each fellow were calculated for each 3-month period. The 85% mark was reached by 4 (31%) of 13 fellows in the first period, 2 of 13 fellows (15%) in the second period, 5 of 11 fellows (45%) in the third period, 7 of 10 fellows (70%) in the fourth period, and 1 of 1 fellow (100%) in the fifth period of training. On the average, it took 7.1 months and 102 ERCPs for trainees to reach desired success levels. Success came more promptly with prior exposure to endoscopy. Fellows without prior endoscopic experience required 148 cases to reach 85% success. Resident surgical experience with major pancreatic resections increased threefold after establishment of the fellowship. CONCLUSIONS: Training in ERCP is possible within the scope of a surgical fellowship in a reasonable length of time and experience. Complication rates remain low even with fellow involvement. Establishment of an ERCP program increases the focus and experience of pancreas surgery in a surgical residency for chief residents.

Characterization of EBV-transformed B-cells established from an individual homozygously mutated (G329A) in the FUT7 alpha1,3-fucosyltransferase gene.

The alpha1,3-fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x (SLe(x)) on human leukocytes. Recently, individuals were characterized carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding this enzyme. The mutated FUT7 construct produced a Fuc-TVII enzyme with impaired activity compared with the wildtype enzyme. Polymorphonuclear granulocytes from an individual carrying this mutation homozygously also showed a reduced expression of SLe(x). In the present study, we have established Epstein-Barr virus-transformed B-cell lines from this individual (SIGN) and from an individual not carrying the mutation (IWO). The cell lines were confirmed to be of B-cell origin by flow cytometry analysis. IWO cells interacted with E-selectin in an in vitro flow chamber analysis whereas SIGN cell did not. However, when SIGN cell was transiently transfected with wildtype FUT7 cDNA, interaction with E-selectin could be restored. Cell surface expression of the SLe(x)-related epitopes recognized by antibodies CSLEX-1, KM-93 and HECA-452 was elevated on IWO cells compared with that on SIGN cells, consistent with a role of these antigens in E-selectin recognition. These cell lines will be useful in further characterization of E-selectin ligands and encourage further studies on the consequences of the FUT7-G329A mutation in vivo.

Role of pancreatic duct stenting in the treatment of chronic pancreatitis.

BACKGROUND: Endoscopic retrograde cholangiopancreatography and stent placement are relatively new alternatives to surgery for the treatment of chronic pancreatitis. The objective of this study was to determine the efficacy of pancreatic duct stent placement for the treatment of chronic pancreatitis. METHODS: This study included 89 patients treated with pancreatic stents between 1993 and 2002. The patients were contacted via telephone for a personal interview with regard to pain, medication usage, weight loss or gain, and eating patterns. Additionally, medication usage before and after treatment was documented from the Kentucky Cabinet for Health Services' electronic reporting system for narcotic use. RESULTS: Of the 89 patients, 9 were deceased, 5 either refused to interview or could not be contacted, and 75 were interviewed. Significant weight gain exceeding 15 lb after treatment was experienced by 22%, whereas only 4% lost weight. A majority of the patients (68%) noted that they had less severe relapses or no relapses after treatment. The patients reported a decrease in pain level on a 10-point scale from 8.7 to 4.1 (53% decrease) after treatment. A decrease in pain medication usage was reported by 47% of the patients, and 83% considered their treatment successful. The Kentucky All Schedule Prescription Electronic Report (KASPER) was obtained before and after treatment for 55 patients. According to this statewide electronic reporting system, 63% had a documented decrease in narcotic use. CONCLUSION: The findings of this study support the use of pancreatic duct stenting as an option before surgical intervention for these difficult-to-manage patients with chronic pancreatitis.

Stabilizing feedbacks in glacier-bed erosion.

Glaciers often erode, transport and deposit sediment much more rapidly than nonglacial environments, with implications for the evolution of glaciated mountain belts and their associated sedimentary basins. But modelling such glacial processes is difficult, partly because stabilizing feedbacks similar to those operating in rivers have not been identified for glacial landscapes. Here we combine new and existing data of glacier morphology and the processes governing glacier evolution from diverse settings to reveal such stabilizing feedbacks. We find that the long profiles of beds of highly erosive glaciers tend towards steady-state angles opposed to and slightly more than 50 per cent steeper than the overlying ice-air surface slopes, and that additional subglacial deepening must be enabled by non-glacial processes. Climatic or glaciological perturbations of the ice-air surface slope can have large transient effects on glaciofluvial sediment flux and apparent glacial erosion rate.

Laparoscopic choledochojejunostomy and gastrojejunostomy in a porcine model.

BACKGROUND: Surgical extirpation remains the only known curative treatment for cancer of the pancreas. Because of locally advanced or metastatic tumor, up to 80% of patients are unresectable at the time of initial diagnosis [13]. Other investigators previously have suggested that laparoscopy before laparotomy aids in the diagnosis of unresectable pancreatic cancer in a fair number of patients even after negative computed tomography scans [3, 17]. Many surgeons are reluctant to incorporate laparoscopy into the workup of patients with cancer of the pancreas because of the frequent need for surgical bypass in the management of either biliary tract obstruction or gastric outlet obstruction [9, 13]. Previous studies have demonstrated the feasibility of laparoscopic cholecystojejunostomy combined with gastrojejunostomy in a porcine model, as well as the individual accomplishment of laparoscopic choledochojejunostomy. The purpose of this study was to document the feasibility of performing laparoscopic choledochojejunostomy with gastrojejunostomy. METHODS: Under general anesthesia, seven pigs underwent laparoscopic choledochojejunostomy and gastrojejunostomy using an intracorporeal hand-sutured technique. RESULTS: The mean operating time ranged from 150 to 450 min. All the animals recovered completely from the operation and had patent anastomoses at the time of necropsy. One pig died of gastric bleeding on postoperative day 13, and two animals had intraabdominal fluid collections discovered at the time of necropsy. CONCLUSIONS: These results suggest that synchronous laparoscopic bypass of biliary and gastric outlet obstruction is feasible, and can be performed in a manner similar to that used in open operations. We believe this lends support to the argument promoting laparoscopy in the evaluation of pancreatic cancer.

Factor V Leiden (G1691A) and prothrombin gene G20210A mutations as potential risk factors for venous thromboembolism after total hip or total knee replacement surgery.

Patients (n = 1600) from 12 European countries, scheduled for elective orthopaedic hip or knee surgery, were screened for Factor V Leiden and prothrombin gene G20210A mutations, found in 5.5% and 2.9% of the populations, respectively. All patients underwent prophylactic treatment with one of four doses of melagatran and ximelagatran or dalteparin, starting pre-operatively. Bilateral ascending venography was performed on study day 8-11. The patients were subsequently treated according to local routines and followed for 4-6 weeks postoperatively. The composite endpoint of screened deep vein thrombosis (DVT) and symptomatic pulmonary embolism (PE) during prophylaxis did not differ significantly between patients with or without these mutations. Symptomatic venous thromboembolism (VTE) during prophylaxis and follow-up (1.9%) was significantly over-represented among patients with the prothrombin gene G20210A mutation (p = 0.0002). A tendency towards increased risk of VTE was found with the Factor V Leiden mutation (p = 0.09). PE were few, but significantly over-represented in both the Factor V Leiden and prothrombin gene G20210A mutated patients (p = 0.03 and p = 0.05, respectively). However, since 90% of the patients with these genetic risk factors will not suffer a VTE event, a general pre-operative genotyping is, in our opinion, of questionable value.

Cost comparison of endoscopic stenting vs surgical treatment for unresectable cholangiocarcinoma.

BACKGROUND: Total lifetime costs of endoscopic vs surgical treatment for obstructive jaundice due to cholangiocarcinoma are difficult to assess. The purpose of this study was to compare total costs in these two groups, including all treatment and retreatments. METHODS: This retrospective study identified patients with biopsy-proven cholangiocarcinoma treated this decade with either endoscopic biliary stenting or surgical biliary-enteric bypass with or without resection. Outcomes and hospital charges were recorded. Ten matched control patients were compared from each group. Costs included those for cost of repeat endoscopy in the endoscopic group and for the management of recurrent obstructive jaundice in the surgical group. RESULTS: The groups were similar in age and gender ratio. The surgical approach was frequently for cure; therefore, surgical patients were by and large at an earlier stage of their disease than those in the endoscopic group. Mean survival for the endoscopic group was 19 months vs 16.5 months for the surgical group. The median total lifetime cost for surgical therapy was $60,986 vs $24,251 for endoscopic therapy. CONCLUSION: Endoscopic therapy is an effective palliative therapy for unresectable cholangiocarcinoma. Endoscopic therapy for this entity costs significantly less than surgical treatment. The cost benefit of endoscopic stenting over standard surgical bypass in the management of patients with unresectable cholangiocarcinoma, when considered along with its minimally invasive approach, makes this the procedure of choice for palliative therapy.

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.

Determination of Lewis FUT3 gene mutations by PCR using sequence-specific primers enables efficient genotyping of clinical samples.

We have developed a polymerase chain reaction method using sequence-specific primers (PCR-SSP) for rapid and correct genotyping of the common Lewis (FUT3) gene mutations 59T>G, 202T>C, 314C>T, 508G>A, and 1067T>A. The PCR-SSP method was validated on 20 healthy blood donors and 16 non-insulin-dependent diabetic patients. All individuals were in parallel genotyped by our established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The FUT3 genotypes, determined with the PCR-SSP method, were in complete accordance with the results of the PCR-RFLP reference method. The PCR-SSP method could also be adapted to assign the presence of a specific mutation to the respective FUT3 alleles. We found the method to be reliable, rapid and cheap with no requirements for restriction enzyme processing.

Incorporation of selegiline metabolites into hair after oral selegiline intake.

We have previously shown that melanin in human hair has a great impact on the incorporation of codeine into hair. The present study on 10 subjects was performed to investigate whether or not these findings could also be extrapolated to other therapeutic drugs. We chose selegiline because it metabolizes to two commonly abused central stimulants, methamphetamine and amphetamine. The results would therefore also be of interest when studying the intake of such drugs and their incorporation into human hair. Selegiline and metabolites were determined by gas chromatography-mass spectrometry, total melanin by spectrophotometry, and pyrrole-tricarboxylic acid by high-performance liquid chromatography with ultraviolet detection. Our results show strong positive exponential relationships (y = e(x)) between melanin and the metabolites, which for methamphetamine improved by normalizing for plasma area under the curve. We conclude that the major metabolites of selegiline can be detected in hair up to four weeks after a single oral dose and that the incorporation closely relates to the melanin contents.

Physical symptoms as indicators of depression and anxiety.

Psychological problems are the single most common reason for attrition from U.S. Navy basic training. A contributing factor is that, although military entrance processing station physicians assess numerous aspects of physical health, there is little rigor behind attempts to identify psychological disorders. In the present study, we highlight previous research indicating that patterns of physical health symptoms can provide a gauge of psychological dysfunction. We also report significant relationships between physical symptoms, anxiety, and depression in a sample of more than 2,000 sailors. Therefore, given the links between physical symptoms and psychological problems, it may be feasible for physicians who screen military applicants to use routinely gathered physical health information as part of an improved system to detect psychological disorders.