Effect of historical factors on genetic variation in three terrestrial Cephalanthera species (Orchidaceae) with different breeding system on the Korean Peninsula

Previous studies have shown that levels of genetic diversity in species of the genus Cephalanthera covary with the breeding system. In the southern part of the Korean Peninsula, the three self‐compatible terrestrial orchids Cephalanthera erecta, C. falcata and C. longibracteata flower synchronously in sympatric populations. The food‐deceptive C. falcata with bright yellow flowers is predominantly outcrossing, whereas autogamy is the dominant strategy in both C. erecta and C. longibracteata, whose white flowers do not open fully. We examined genetic diversity (by means of allozymes) of the three species in sympatric populations (600 × 600 m area) in the Yeonwhasan Provincial Park (YPP) and in non‐sympatric populations outside YPP, South Korea. Thirteen out of 20 putative loci were variable across the three species, but there was a complete lack of allozyme variation within each species and we found no evidence of hybridisation. Our results suggest that historical factors, i.e. the Quaternary climate oscillations, have played a major role in determining levels of genetic diversity in the three Cephalanthera species. The Korean populations of C. erecta (a warm‐temperate/temperate element) and C. falcata (a warm‐temperate element) may have been established by a single introduction from a genetically depauperate ancestral population, likely located outside the Korean Peninsula. On the other hand, since C. longibracteata is a boreal/temperate element, it may have survived the Last Glacial Maximum in microrefugia located in low elevation regions within the Peninsula where it has been subjected to population bottlenecks reducing its genetic diversity.     

Lantana camara Essential Oils from Vietnam: Chemical Composition,Molluscicidal, and Mosquito Larvicidal Activity

Lantana camara is a troublesome invasive plant introduced to many tropical regions, including Southeast Asia. However, the plant does hold promise as a source of essential oils that may be explored for potential use. Fresh water snails such as Pomacea canaliculata, Gyraulus convexiusculus, and Tarebia granifera can be problematic agricultural pests as well as hosts for parasitic worms. Aedes and Culex mosquitoes are notorious vectors of numerous viral pathogens. Control of these vectors is of utmost importance. In this work, the essential oil compositions, molluscicidal, and mosquito larvicidal activities of four collections of L. camara from north-central Vietnam have been investigated. The sesquiterpene-rich L. camara essential oils showed wide variation in their compositions, not only compared to essential oils from other geographical locations (at least six possible chemotypes), but also between the four samples from Vietnam. L. camara essential oils showed molluscicidal activities comparable to the positive control, tea saponin, as well as other botanical agents. The median lethal concentrations (LC₅₀) against the snails were 23.6–40.2 μg/mL (P. canaliculata), 7.9–29.6 μg/mL (G. convexiusculus), and 15.0–29.6 μg/mL (T. granifera). The essential oils showed good mosquito larvicidal activities with 24-h LC₅₀ values of 15.1–29.0 μg/mL, 26.4–53.8 μg/mL, and 20.8–59.3 μg/mL against Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus, respectively. The essential oils were more toxic to snails and mosquito larvae than they were to the non-target water bug, Diplonychus rusticus (24-h LC₅₀=103.7–162.5 μg/mL). Sesquiterpene components of the essential oils may be acting as acetylcholinesterase (AChE) inhibitors. These results suggest that the invasive plant, L. camara, may be a renewable botanical pesticidal agent.     

Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis

Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr¹⁷⁶, but not Trp²³¹, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr¹⁷⁶ as the anchoring residue. Importantly, mutation of TWEAK at Tyr¹⁷⁶ did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.     

A multi-center randomised controlled trial of gatifloxacin versus azithromycin for the treatment of uncomplicated typhoid fever in children and adults in Vietnam

Background

Drug resistant typhoid fever is a major clinical problem globally. Many of the first line antibiotics, including the older generation fluoroquinolones, ciprofloxacin and ofloxacin, are failing.

Objectives

We performed a randomised controlled trial to compare the efficacy and safety of gatifloxacin (10 mg/kg/day) versus azithromycin (20 mg/kg/day) as a once daily oral dose for 7 days for the treatment of uncomplicated typhoid fever in children and adults in Vietnam.

Methods

An open-label multi-centre randomised trial with pre-specified per protocol analysis and intention to treat analysis was conducted. The primary outcome was fever clearance time, the secondary outcome was overall treatment failure (clinical or microbiological failure, development of typhoid fever-related complications, relapse or faecal carriage of S. typhi).

Principal Findings

We enrolled 358 children and adults with suspected typhoid fever. There was no death in the study. 287 patients had blood culture confirmed typhoid fever, 145 patients received gatifloxacin and 142 patients received azithromycin. The median FCT was 106 hours in both treatment arms (95% Confidence Interval [CI]; 94–118 hours for gatifloxacin versus 88–112 hours for azithromycin), (logrank test p = 0.984, HR [95% CI] = 1.0 [0.80–1.26]).Overall treatment failure occurred in 13/145 (9%) patients in the gatifloxacin group and 13/140 (9.3%) patients in the azithromycin group, (logrank test p = 0.854, HR [95% CI] = 0.93 [0.43–2.0]). 96% (254/263) of the Salmonella enterica serovar Typhi isolates were resistant to nalidixic acid and 58% (153/263) were multidrug resistant.

Conclusions

Both antibiotics showed an excellent efficacy and safety profile. Both gatifloxacin and azithromycin can be recommended for the treatment of typhoid fever particularly in regions with high rates of multidrug and nalidixic acid resistance. The cost of a 7-day treatment course of gatifloxacin is approximately one third of the cost of azithromycin in Vietnam.

Trial Registration

Controlled-Trials.com ISRCTN67946944     

Maintenance of Chronic Fatigue Syndrome (CFS) in Young CFS Patients Is Associated with the 5-HTTLPR and SNP rs25531 A > G Genotype

Earlier studies have shown that genetic variability in the SLC6A4 gene encoding the serotonin transporter (5-HTT) may be important for the re-uptake of serotonin (5-HT) in the central nervous system. In the present study we investigated how the 5-HTT genotype i.e. the short (S) versus long (L) 5-HTTLPR allele and the SNP rs25531 A > G affect the physical and psychosocial functioning in patients with chronic fatigue syndrome (CFS). All 120 patients were recruited from The Department of Paediatrics at Oslo University Hospital, Norway, a national referral center for young CFS patients (12–18 years). Main outcomes were number of steps per day obtained by an accelerometer and disability scored by the Functional Disability Inventory (FDI). Patients with the 5-HTT SS or SL_G genotype had a significantly lower number of steps per day than patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. Patients with the 5-HTT SS or SL_G genotype also had a significantly higher FDI score than patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. Thus, CFS patients with the 5-HTT SS or SL_G genotype had worse 30 weeks outcome than CFS patients with the 5-HTT L_AL_G, SL_A or L_AL_A genotype. The present study suggests that the 5-HTT genotype may be a factor that contributes to maintenance of CFS.     

Risk factors of Streptococcus suis infection in Vietnam. A case-control study

Background

Streptococcus suis infection, an emerging zoonosis, is an increasing public health problem across South East Asia and the most common cause of acute bacterial meningitis in adults in Vietnam. Little is known of the risk factors underlying the disease.

Methods and Findings

A case-control study with appropriate hospital and matched community controls for each patient was conducted between May 2006 and June 2009. Potential risk factors were assessed using a standardized questionnaire and investigation of throat and rectal S. suis carriage in cases, controls and their pigs, using real-time PCR and culture of swab samples. We recruited 101 cases of S. suis meningitis, 303 hospital controls and 300 community controls. By multivariate analysis, risk factors identified for S. suis infection as compared to either control group included eating “high risk” dishes, including such dishes as undercooked pig blood and pig intestine (OR₁ = 2.22; 95%CI = [1.15–4.28] and OR₂ = 4.44; 95%CI = [2.15–9.15]), occupations related to pigs (OR₁ = 3.84; 95%CI = [1.32–11.11] and OR₂ = 5.52; 95%CI = [1.49–20.39]), and exposures to pigs or pork in the presence of skin injuries (OR₁ = 7.48; 95%CI = [1.97–28.44] and OR₂ = 15.96; 95%CI = [2.97–85.72]). S. suis specific DNA was detected in rectal and throat swabs of 6 patients and was cultured from 2 rectal samples, but was not detected in such samples of 1522 healthy individuals or patients without S. suis infection.

Conclusions

This case control study, the largest prospective epidemiological assessment of this disease, has identified the most important risk factors associated with S. suis bacterial meningitis to be eating ‘high risk’ dishes popular in parts of Asia, occupational exposure to pigs and pig products, and preparation of pork in the presence of skin lesions. These risk factors can be addressed in public health campaigns aimed at preventing S. suis infection.     

Cell migration and invasion assays

The processes of cell migration and invasion are integral to embryonic development and the functioning of adult organisms. Deregulation of these processes contributes to numerous diseases. Ras GTPases and in particular members of the Rho subfamily of GTPases play critical roles in cell migration and invasion. Here, we provide a collection of protocols to assay these functions. We describe two cell migration assays. The monolayer wound healing assay is very easy to implement, whereas the microliter-scale migration assay allows examination of cell behavior on defined extracellular matrices. We also describe two methods that allow the quantification of tumor cell invasion, a versatile transwell Matrigel invasion assay and an organotypic assay that examines the invasion of glioma cells through a rat brain slice.     

Purification of urease from Ureaplasma urealyticum

We have purified urease from the Mollicutes, Ureaplasma urealyticum, using high performance liquid chromatography methods and DEAE-Sephadex chromatography. While only small amounts of material could be utilized in these methods, urease was purified at least 180-fold, yield a major band on SDS-PAGE of 66,000 daltons, a minor band of 64,000 daltons, and several faint bands of lower molecular mass. These results suggest that the 380,000 dalton intact urease is a pentamer or hexamer of these two larger subunits. The highly purified urease from DEAE-Sephadex retained full activity for at least 20 days at 4 degrees C in sodium phosphate buffer (pH 7.2) with 1% bovine serum albumin. The estimated specific activity of the DEAE peak fractions, 180 IU/micrograms, is at least 90-fold greater than that of jack bean urease.     

An Analysis of Skin Test Responses to Spherulin-Based Coccidioidin (Spherusol®) Among a Group of Subjects with Various Forms of Active Coccidioidomycosis

Mycopathologia - A reformulated skin test for coccidioidomycosis, Spherusol®, was recently approved for use in the USA. We hypothesized that it could be useful in predicting severity of...     

Expression of the lysophospholipid receptor family and investigation of lysophospholipid-mediated responses in human macrophages

Some of the biological effects of lipoproteins have been attributed to their association with lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). These lysophospholipids mediate multiple biological responses via several G protein-coupled receptors (GPR). The expression of these receptors, however, has not been systematically investigated in primary human monocytes and macrophages as major cells involved in atherosclerosis. The mRNAs for all 15 receptors described so far were detected in monocytes, macrophages, foam cells and high density lipoprotein (HDL(3))-treated cells using real time RT-PCR. Immunoblots revealed that S1P(1), S1P(2), S1P(4), LPA(1), LPA(2) and GPR65 are expressed in monocytes and macrophages, while S1P(5) and LPA(3) have not been detected. S1P(3) was induced during differentiation but down-regulated by lipid-loading and HDL(3), whereas LPA(1) was down-regulated in differentiated macrophages. The influence of S1P on macrophages was investigated and the induction of CD32 indicates an enhanced phagocytic activity. Altogether, these data give insights into the expression and regulation of lysophospholipid receptors in primary human monocytes, macrophages and foam cells.