Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.

BACKGROUND

Edible plants such as Cratoxylum formosum (Jack) Dyer, Curcumin longa Lin, Momordica charantia Lin and Moringa oleifera Lam have long been believed in Thai culture to relieve ulcers and the symptoms of liver disease. However, little is known about their anti-liver cancer properties and antiviral activity against hepatitis B virus (HBV). The aim of this study was to investigate the anti-liver cancer and anti-HBV activities of crude extracts from these edible plants on human liver cancer cells.

METHODS

Plant samples were prepared and extracted using buffer and hydro-alcoholic solvents. The MTT assay was performed to investigate the effects of the plant extracts on the cell viability of HepG2 cells. The inhibitory effect on replication of HBV was analysed by determining the level of HBV covalently closed circular DNA (cccDNA) in transiently transfected HepG2 cells with the DNA expression plasmid of the HBV genome using a quantitative real-time PCR.

RESULTS

Buffer and hydroalcoholic extracts from C. formosum (leaf) reduced cell viability of HepG2 cells and they also inhibited HBV cccDNA. Crude extracts from C. longa (bulb) in both solvents did not have any cytotoxic effects on the HepG2 cells, but they significantly decreased the level of HBV cccDNA. Buffer extracts from the leaves of M. charantia and the fruits of M. oleifera showed to have anti-HBV activity and also a mild cytotoxicity effect on the HepG2 cells. In addition, leaves of M. Oleifera extracted by hydroalcoholic solvent drastically decreased the level of cccDNA in transiently transfected HepG2 cells.

CONCLUSIONS

Some crude extracts of edible plants contain compounds that demonstrate anti-liver cancer and anti-HBV activities.

Phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the first step in the synthesis of cardiolipin, an acidic phospholipid found in the mitochondrial inner membrane. In the yeast Saccharomyces cerevisiae, PGPS expression is coordinately regulated with general phospholipid synthesis and is repressed when cells are grown in the presence of the phospholipid precursor inositol (M. L. Greenberg, S. Hubbell, and C. Lam, Mol. Cell. Biol. 8:4773-4779, 1988). In this study, we examined the regulation of PGPS in growth conditions affecting mitochondrial development (carbon source, growth stage, and oxygen availability) and in strains with genetic lesions affecting mitochondrial function. PGPS derepressed two- to threefold when cells were grown in a nonfermentable carbon source (glycerol-ethanol), and this derepression was independent of the presence of inositol. PGPS derepressed two- to fourfold as cells entered the stationary phase of growth. Stationary-phase derepression occurred in both glucose- and glycerol-ethanol-grown cells and was slightly greater in cells grown in the presence of inositol and choline. PGPS expression in mitochondria was not affected when cells were grown in the absence of oxygen. In mutants lacking mitochondrial DNA [( rho0] mutants), PGPS activity was 30 to 70% less than in isogenic [rho+] strains. PGPS activity in [rho0] strains was subject to inositol-mediated repression. PGPS activity in [rho0] cell extracts was derepressed twofold as the [rho0] cells entered the stationary phase of growth. No growth phase derepression was observed in mitochondrial extracts of the [rho0] cells. Relative cardiolipin content increased in glycerol-ethanol-grown cells but was not affected by growth stage or by growth in the presence of the phospholipid precursors inositol and choline. These results demonstrate that (i) PGPS expression is regulated by factors affecting mitochondrial development; (ii) regulation of PGPS by these factors is independent of cross-pathway control; and (iii) PGPS expression is never fully repressed, even during anaerobic growth.

Glucomoringin (4(α-L-rhamnosyloxy)-benzyl glucosinolate) (GMG) is an uncommon member of glucosinolate group belonging to the Moringaceae family, of which Moringa oleifera Lam. is the most widely distributed. Bioactivation of GMG with the enzyme myrosinase forms the corresponding isothiocyanate (4(α-L-rhamnosyloxy)-benzyl isothiocyanate) (GMG-ITC), which can play a key role in antitumoral activity and counteract the inflammatory response. The aim of this study was to assess the effect of GMG-ITC treatment in an experimental mouse model of multiple sclerosis (MS), an inflammatory demyelinating disease with neurodegeneration characterized by demyelinating plaques, neuronal, and axonal loss. For this reason, C57Bl/6 male mice were injected with myelin oligodendrocyte glycoprotein35-55 which is able to evoke an autoimmune response against myelin fibers miming human multiple sclerosis physiopatogenesis. Results clearly showed that the treatment was able to counteract the inflammatory cascade that underlies the processes leading to severe MS. In particular, GMG-ITC was effective against proinflammatory cytokine TNF-α. Oxidative species generation including the influence of iNOS, nitrotyrosine tissue expression and cell apoptotic death pathway was also evaluated resulting in a lower Bax/Bcl-2 unbalance. Taken together, this work adds new interesting properties and applicability of GMG-ITC and this compound can be suggested as a useful drug for the treatment or prevention of MS, at least in association with current conventional therapy.

OBJECTIVE

Does physical exercise training improve physical function and quality of life in people with cognitive impairment and dementia? Which training protocols improve physical function and quality of life? How do cognitive impairment and other patient characteristics influence the outcomes of exercise training?

METHODS

Systematic review with meta-analysis of randomised trials.

METHODS

People with mild cognitive impairment or dementia as the primary diagnosis.

METHODS

Physical exercise.

METHODS

Strength, flexibility, gait, balance, mobility, walking endurance, dual-task ability, activities of daily living, quality of life, and falls.

RESULTS

Forty-three clinical trials (n=3988) were included. According to the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system, the meta-analyses revealed strong evidence in support of using supervised exercise training to improve the results of 30-second sit-to-stand test (MD 2.1 repetitions, 95% CI 0.3 to 3.9), step length (MD 5cm, 95% CI 2 to 8), Berg Balance Scale (MD 3.6 points, 95% CI 0.3 to 7.0), functional reach (3.9cm, 95% CI 2.2 to 5.5), Timed Up and Go test (-1second, 95% CI -2 to 0), walking speed (0.13m/s, 95% CI 0.03 to 0.24), and 6-minute walk test (50m, 95% CI 18 to 81) in individuals with mild cognitive impairment or dementia. Weak evidence supported the use of exercise in improving flexibility and Barthel Index performance. Weak evidence suggested that non-specific exercise did not improve dual-tasking ability or activity level. Strong evidence indicated that exercise did not improve quality of life in this population. The effect of exercise on falls remained inconclusive. Poorer physical function was a determinant of better response to exercise training, but cognitive performance did not have an impact.

CONCLUSIONS

People with various levels of cognitive impairment can benefit from supervised multi-modal exercise for about 60minutes a day, 2 to 3days a week to improve physical function. [Lam FMH , Huang MZ, Liao LR, Chung RCK, Kwok TCY, Pang MYC (2018) Physical exercise improves strength, balance, mobility, and endurance in people with cognitive impairment and dementia: a systematic review. Journal of Physiotherapy 64: 4-15].

Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (AmLAM) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of AmLAM follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent AmLAM expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality.

BACKGROUND

Because about 50% of the Zimbabwean population is at risk of contracting malaria each year, the majority of people, especially in rural areas, use traditional plant-based medicines to combat malaria. This explorative ethnobotanical survey was undertaken to document how malaria is conceptualized and diagnosed by traditional healers, and to record the medicinal plants used in the prevention and treatment of malaria, their mode of preparation and administration.

METHODS

The research was conducted in three villages in Headman Muzite׳s area and in Chiriga village. These villages are located in the Chipinge district in the Manicaland Province in Zimbabwe.Traditional healers were selected with the assistance of the headman of the Muzite area and a representative of the Zimbabwe National Traditional Healers Association. Semi-structured interviews were conducted with 14 traditional healers from four villages in the Chipinge district in Zimbabwe.

RESULTS

In total, 28 plants from 16 plant families are used by the healers who manage malaria with medicinal plants. The most cited plant is Cassia abbreviata Oliv. (Leguminosae) followed by Aristolochia albida Duch (Aristolociaceae) and Toddalia asiatica (L.) Lam. (Rutaceae). Roots (55.3%) are the most common part used. Most of the plant parts used to treat malaria are stored as dried powders in closed bottles. The powders are soaked in hot or cold water and the water extract is taken as the active medicine. The healers consider their medicinal knowledge as a spiritual family heritage. Only 25% of the healers refer the malaria patients that do not respond to their treatment to hospital - they believe evil spirits cause their remedies to failure and they would rather try a different plant or perform a cleansing ceremony.

CONCLUSIONS

Local knowledge of medicinal plants in the treatment of malaria still exists in all four villages surveyed and traditional healers appear to play an important role in primary health care services in this remote rural area in Zimbabwe. This explorative survey underscores the need to preserve and document traditional healing for managing malaria and for more future scientific research on the plants to determine their efficacy and their safety. This could improve their traditional anti-malarial recipes and might contribute to a better integration of Zimbabwean traditional medicine into the national health system in the future.

Due to increasing mupirocin resistance, alternatives for Staphylococcus aureus nasal decolonization are needed. Lauric acid monoesters combined with lactic, mandelic, malic, or benzoic acid are being evaluated as possible alternatives. We determined the in vitro activity of 13 lauric acid monoester (<em>LAM</em>) formulations and mupirocin against 30 methicillin-susceptible S. aureus (MSSA) isolates and 30 methicillin-resistant S. aureus (MRSA) isolates. We then used a murine model of MRSA nasopharyngeal colonization to compare the in vivo activity of mupirocin with three <em>LAM</em> formulations. MSSA and MRSA MIC(90) values were 0.25 microg/ml for mupirocin and </=4 microl/ml for all <em>LAM</em> formulations tested. Hsd:ICR mice were challenged with 10(8) CFU/naris MRSA. Five days later, S. aureus colonization was documented by culture. Treatment with bland, mupirocin, or one of three <em>LAM</em> ointments was then administered unblinded thrice daily for 2 days. Three days after treatment, both anterior nares were cultured for S. aureus. Administration of 128774-49E or 128774-53A was associated with greater eradication of MRSA carriage (24/34 [71%] or 33/40 [83%]) of animals, respectively) than bland ointment (12/38 [32%]) (P < 0.005). 128774-53A administration resulted in greater MRSA carriage eradication than mupirocin (19/38 [50%]) (P < 0.005) in this model. <em>LAM</em> formulations warrant evaluation for S. aureus nasal decolonization in humans.

The spoligotyping-based population structure of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated in Poland (n = 46), representing all culture-positive MDR tuberculosis (MDR-TB) cases, was compared to that of isoniazid (INH)-monoresistant strains (n = 71) isolated in 2004. The latter data set from a previous study (E. Augustynowicz-Kopeć, T. Jagielski, and Z. Zwolska, J. Clin. Microbiol. 2008, 46:4041-4044) represented 87% of all INH-monoresistant strains. The clustering rates and genotypic-diversity indexes for the 2 subpopulations were not significantly different (P = 0.05). The results were entered in the SITVIT2 database to assign specific shared type designations, corresponding genotypic lineages, and geographical distributions and compared to available data from neighboring countries (Germany, n = 704; Czech Republic, n = 530; Sweden, n = 379; Kaliningrad, Russia, n = 90) and strains from previous studies in Poland (n = 317). MDR strains resulted in 27 patterns (20 unique strains within the study and 7 clusters containing 2 to 6 isolates per cluster with a clustering rate of 56.5%) and belonged to the following genotypic lineages: ill-defined T family (28.3%), Haarlem (17.4%), Latin American and Mediterranean (LAM) (13%), Beijing (8.7%), S family (4.35%), and the X clade (2.17%). Comparison of the genetic structure of the MDR strains with that of INH-monoresistant strains showed that a total of 9 patterns were shared by both groups; these represented 1/3 of the MDR strains and 2/3 of the INH-monoresistant strains. Interestingly, 76.1% of the MDR isolates and 71.8% of the INH-resistant isolates yielded spoligotypes that were previously reported from Poland. The observation that nearly half of the spoligotypes identified among both MDR (48.1%) and INH-monoresistant (43.3%) M. tuberculosis isolates were present in Poland's neighboring countries suggested that a significant proportion of MDR and INH-resistant TB cases in Poland were caused by strains actively circulating in Poland or its neighbors. Our results corroborate the leading role of the T and Haarlem genotypes in the epidemiology of drug-resistant TB in Poland. Nevertheless, the LAM and Beijing family strains that infected, correspondingly, 13% and 9% of patients with MDR-TB were absent among the strains from patients with INH-monoresistant TB, suggesting that a proportion of MDR-TB cases in Poland are due to ongoing transmission of MDR clones exhibiting specific genotypes. Study of the population genetic relationships between MDR and INH-monoresistant strains by drawing minimum spanning trees showed that ill-defined T1 sublineage strains (1/3 of all INH-monoresistant strains), represented by its prototype, SIT53, constituted the central node of the tree, followed by strains belonging to the well-defined H3, H1, and S subgroups. However, the MDR group, in addition, contained LAM (n = 6) and Beijing (n = 4) lineage isolates. With the exception of the 4 Beijing lineage strains in the latter group and a single orphan isolate in the INH-monoresistant group, none of the remaining 112/117 isolates belonged to principal genetic group 1 (PGG1) in our study. Given the high rate of clustering and the near absence of immigrants in the study, the persistence of MDR-TB in Poland seems to result from active transmission of MDR strains within the autochthonous population, the bulk of it caused by evolutionarily recent tubercle bacilli.

Mycobacteria are major human pathogens responsible for such serious and widespread diseases as tuberculosis and leprosy. Among the evolutionary adaptations essential for pathogenicity in mycobacteria is a complex carbohydrate-rich cell-wall structure that contains as a major immunomodulatory molecule the polysaccharide lipoarabinomannan (LAM). We report here crystal structures of three fragments from the non-reducing termini of LAM in complex with a murine antibody Fab fragment (CS-35Fab). These structures reveal for the first time the three-dimensional structures of key components of LAM and the molecular basis of LAM recognition at between 1.8- and 2.0-A resolution. The antigen-binding site of CS-35Fab forms three binding pockets that show a high degree of complementarity to the reducing end, the branch point and one of the non-reducing ends of the Y-shaped hexasaccharide moiety found at most of the non-reducing termini of LAM. Structures of CS-35Fab bound to two additional tetrasaccharides confirm the general mode of binding seen in the hexasaccharide and indicate how different parts of LAM are recognized. Altogether, these structures provide a rational basis for understanding the overall architecture of LAM and identify the key elements of an epitope that may be exploited for the development of novel and more effective anti-mycobacterial vaccines. Moreover, this study represents the first high-resolution X-ray crystallographic investigation of oligofuranoside-protein recognition.

Leafy vegetables [Basella rubra L., Peucedanum sowa Roxb., Moringa oleifera Lam., Trigonella foenum-graecum L., Spinacia oleracea L., Sesbania grandiflora (L.) Poir., and Raphanus sativus L.] that are commonly used by the rural population in India were evaluated in terms of their main carotenoid pattern. The extracted carotenoids were purified by open column chromatography (OCC) on a neutral alumina column to verify their identity by their characteristic UV-visible absorption spectra. Reverse-phase high-performance liquid chromatography (HPLC) on a C18 column with UV-visible photodiode array detection under isocratic conditions was used for quantification of isolated carotenoids. Acetonitrile/methanol/dichloromethane (60:20:20 v/v/v) containing 0.1% ammonium acetate was used as a mobile phase. The major carotenoids identified by both methods were lutein, beta-carotene, violaxanthin, neoxanthin, and zeaxanthin. Among the carotenoids identified, lutein and beta-carotene levels were found to be higher in these leafy vegetables. Results show that P. sowa and S. oleracea are rich sources of lutein (77-92 mg/100 g of dry wt) and beta-carotene (36-44 mg/100 g of dry wt) compared with other leafy vegetables. The purity of carotenoids eluted by OCC was clarified by HPLC, and they were found to be 92% +/- 3% for neoxanthin, 94% +/- 2% for violaxanthin, 97% +/-2% for lutein and zeaxanthin, and 90% +/- 3% for beta-carotene. It could be recommended to use P. sowa and S. oleracea as rich sources of lutein and beta-carotene for health benefits. The OCC method proposed is relatively simple and provides purified carotenoids for feeding trials.

Recent studies of retroviral-mediated gene transfer have shown that retroviral integrations themselves may trigger nonmalignant clonal expansion of hematopoietic stem cells (HSCs) in transplant recipients. These observations suggested that previous conclusions of HSC dynamics based on gamma-retroviral gene marking should be confirmed with improved vectors having a more limited capacity to transactivate endogenous genes. Because of the low trans-activation activity of self-inactivating lentiviral vectors (LVs), we have investigated whether the LV marking of mouse HSCs induces a competitive repopulation advantage in recipients of serially transplants. As deduced from analyses conducted in primary and secondary recipients, we concluded that lentivirally transduced HSCs have no competitive repopulation advantages over untransduced HSCs. By linear amplification-mediated polymerase chain reaction (LAM-PCR) analysis, we characterized LV-targeted genes in HSC clones that engrafted up to quaternary recipients. Although 9 clones harbored integrations close to defined retroviral insertion sites, none was characterized as a common integration site, and none was present in HSC clones repopulating quaternary recipients. Taken together, our results show unaltered repopulation properties of HSCs transduced with LVs, and confirm early studies suggesting the natural capacity of a few HSC clones to generate a monoclonal or oligoclonal hematopoiesis in transplant recipients.

Melatonin (MT) is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [Ipomoea batatas (L.) Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K+/Na+ homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K+/Na+ homeostasis in sweet potato seedlings as indicated by the low reduced K+ content in tissues and low accumulation of Na+ content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K+ efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM) H+-ATPase activity and intracellular adenosine triphosphate (ATP) level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG) accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA) β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H+-ATPase activity, and K+/Na+ homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H+-ATPase activity and K+/Na+ homeostasis in sweet potato.

Experimental data indicate that hippocampal lesions might impair, spare, or even facilitate latent inhibition (LI). Furthermore, when LI is impaired by the lesions, it might be reinstated by haloperidol administration. The present article applies a neural network model of classical conditioning (N. A. Schmajuk, Y. W. Lam, & J. A. Gray, 1996) to investigate the possible causes of these puzzling results. According to the model, LI is manifested because preexposure of the conditioned stimulus (CS) reduces Novelty, defined as proportional to the sum of the mismatches between predicted and observed events, thereby reducing attention to the CS and retarding conditioning. It is assumed that hippocampal lesions affect the prediction of events. Computer simulations reveal that, depending on the behavioral protocol (i.e., procedure and total time of CS preexposure), Novelty in hippocampal lesioned animals might be larger, equal, or smaller (corresponding to smaller, equal, or larger LI) than in normal controls. Reinstatement of LI by haloperidol administration is explained by assuming that dopaminergic antagonists decrease the value of Novelty, when Novelty increases following hippocampal lesions.

Lymphangioleiomyomatosis (LAM) is a rare idiopathic disease predominately affecting women. The disease is characterized by peribronchial, perivascular, and perilymphatic proliferation of smooth muscle like cells resulting in vascular and airway obstruction and cyst formation. The natural history of pulmonary LAM is unknown, and it displays remarkable diversity in its clinical course. More than 400 citations are currently listed in the literature for LAM. This article reviews and updates the rapidly expanding knowledge about LAM.

We sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of ∼3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ≥0.5% of UDPS reads in a sample. Sanger sequencing identified ≥1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%; P = 0.06). UDPS detected ≥1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%; P = 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P < 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

Emergence of a lamivudine (LAM)-resistant hepatitis B virus (HBV) with amino acid substitutions in the YMDD motif is a well-documented problem during long-term LAM therapy. Entecavir (ETV) is a new drug approved for treatment of HBV infection with or without LAM-resistant mutants. This report describes an ETV-resistant strain of HBV, which emerged after prolonged ETV therapy in a patient who did not respond to LAM therapy. Direct sequence analysis of the ETV-resistant strain showed appearance of amino acid substitution rtS202G in the reverse transcriptase (RT) domain, together with rtL180M + M204V substitution that had developed at the emergence of LAM-resistant mutant. In vitro analysis demonstrated that the rtL180M + M204V + S202G mutant strain displayed a 200-fold and a 5-fold reduction in susceptibility to ETV compared with the wild- type and the rtL180M + M204V mutant strain, respectively. Adefovir was effective against the ETV-resistant strain both in vitro and during the clinical course. In conclusion, this study showed that virological and biochemical breakthrough due to ETV could occur in patients infected with LAM-resistant HBV and confirmed that the addition of rtS202G substitution to the rtL180M + M204V mutant strain is responsible for ETV resistance and we could treat the resistant mutant successfully.

Patients with acute cholecystitis sometimes require placement of percutaneous cholecystostomy catheters, either as a bridge to surgery or as primary therapy. In patients who cannot undergo surgery, subsequent removal of the catheter can lead to recurrence of cholecystitis, whereas leaving the drain in place can cause adverse events. We investigated internalization of percutaneous cholecystostomy drainage catheters, using endoscopic ultrasound (EUS)-guided placement of lumen-apposing metal stents (LAMS) as an alternative treatment strategy. Seven patients (median age, 57 years; 6 men) underwent EUS-guided cholecystoenterostomy for internalization of gallbladder drainage with EUS-guided placement of a 10- or 15-mm LAMS. All had initially been treated with placement of a percutaneous cholecystostomy catheter for cholecystitis and were later deemed unfit for cholecystectomy. Technical success was achieved in all patients in 1 endoscopic session, with subsequent removal of all percutaneous drains. Two patients required placement of self-expandable metal stents within the LAMS to successfully bridge the gallbladder and gastrointestinal lumen. No adverse events occurred after a median follow-up of 2.5 months. EUS-guided cholecystoenterostomy using a LAMS is therefore a viable option for internal gallbladder drainage in patients who have a percutaneous cholecystostomy catheter and are poor candidates for cholecystectomy.

We have previously demonstrated that there is a subpopulation of loosely adherent pulmonary mononuclear cells that can be isolated from minced and enzyme-digested lung tissue with a potent capacity to stimulate allogeneic T lymphocyte proliferation. We now demonstrate that these cells are also capable of stimulating an autologous mixed leukocyte reaction (AMLR) and presenting antigen to autologous T lymphocytes. These loosely adherent mononuclear cells (LAM) were more effective than either alveolar macrophages or monocytes as antigen-presenting cells. Depletion of phagocytic or Fc receptor-positive cells from the LAM population enhanced the stimulation of an reaction AMLR while preserving antigen-induced T lymphocyte proliferation. These results indicate that there are nonphagocytic, Fc receptor-negative accessory cells in human lung parenchyma capable of activating resting T cells in an AMLR and supporting antigen-specific T lymphocyte proliferation. The identity of these cells is uncertain, but the data strongly suggest that the cell is not a classical monocyte-derived macrophage. These antigen-presenting cells may be critical in the initiation of immune responses within the lung.

Hepatic oval cells (OC) are considered to be facultative liver stem cells and, because they may undergo differentiation into a variety of cell lineages, they might have the potential to be used in cellular therapy. Signals delivered by extracellular matrix (ECM) proteins take part in cellular differentiation in cooperation with signals from growth factors; indeed, some ECM proteins, such as laminin (LAM) and fibronectin (FN), have been shown to contribute to beta-cell differentiation and islet development, respectively. Since no previous studies have investigated the effect of ECM proteins on the expression of islet cell markers by cultured OC, the purpose of the present study was to evaluate whether FN and LAM modulate the expression of genes related to the endocrine pancreas in these liver cells. OC proliferation was induced in Wistar rats by prolonged treatment with 2-acetoaminofluorene/allyl alcohol and confirmed by reverse transcription/polymerase chain reaction and hepatic immunocytochemical and histopathological analyses. OC isolation was performed by Ficoll gradient and magnetic-activated cell sorting. OC were cultured for 1 and 2 months under several conditions with specific growth factors, over a FN or LAM substrate or in high glucose, nicotinamide and fetal calf serum. OC cultured on FN substrate expressed Pdx-1, Pax-6, insulin 2 and glucagon. LAM also induced the expression of Pdx-1, insulin 1 and insulin 2, glucagon, somatostatin and GLUT-2. Our results suggest that these ECM proteins can be used in protocols of OC transdifferentiation aimed at reducing the period necessary for complete transdifferentiation.

Dried flowers and leaves of four different Achillea species grown wild in several provinces of Iran, including one species collected from three different locations of Isfahan province were analyzed for the headspace volatile components, total phenolics (TP) and tartaric esters (TE). Capillary gas chromatography/mass spectrometry (GC/MS) combined with a purge and trap method was used for quantification of aroma components. Over 70 compounds were determined in the samples. Flower samples from all species contained 2-methyl butanal, alpha-pinene, alpha-thujene, camphene, hexanal, beta-pinene and 1,8-cineole; however, the major constituents of the aerial parts were determined as alpha-pinene, camphene, DL-limonene and 1,8-cineole. The largest number of aroma components were found in Achillea tenuifolia Lam. and Achillea millefolium L. In all species, except A. millefolium, the leaves contained more TP and TE than flowers. However, A. wilhelmsii from Semirom in Isfahan province showed the highest values for TP and TE.

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF(33-42)) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF(33-42) significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P < 0.0001). The agonist peptide (Lam beta 1(925-933)) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P < 0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis.

Mechanistic target of rapamycin inhibitors reduce loss of lung function in lymphangioleiomyomatosis (LAM), although their benefit varies between individuals. We examined lung function response and side effects to rapamycin in a national cohort.

Subjects were receiving rapamycin for progressive lung disease. Clinical evaluation, detailed phenotyping, serial lung function, rapamycin and safety monitoring were performed according to a clinical protocol. Lung function change, measured as FEV1 slope (ΔFEV1), was reported for those treated for 1 year or longer.

Rapamycin was associated with improved ΔFEV1 in 21 individuals where pretreatment data were available (p<0.0001). In 47 treated for a mean duration of 35.8 months, mean ΔFEV1 was +11 (SD 75) mL/year, although it varied from +254 to -148 mL/year. The quartile with the highest positive ΔFEV1 had greater pretreatment FEV1 (p=0.02) and shorter disease durations (p=0.02) than the lowest quartile. Serum rapamycin level was positively associated with side effects (p=0.02) but not ΔFEV1 over 1 year. Within the first month of therapy, apthous ulcers, nausea and diarrhoea were associated with higher rapamycin levels. Acne, oedema and menstrual irregularities tended to increase over the first year of therapy. At the end of observation, the prevalence of side effects was 5% or less.

Rapamycin reduces lung function loss in LAM, although in some, ΔFEV1 continues to fall at an accelerated rate. Poor response to rapamycin was associated with lower pretreatment lung function and longer disease duration but not serum level. Early intervention with low-dose rapamycin may preserve lung function and reduce side effects.

Moringa oleifera Lam., also known as the 'drumstick tree,' is recognized as a vibrant and affordable source of phytochemicals, having potential applications in medicines, functional food preparations, water purification, and biodiesel production. The multiple biological activities including antiproliferation, hepatoprotective, anti-inflammatory, antinociceptive, antiatherosclerotic, oxidative DNA damage protective, antiperoxidative, cardioprotective, as well as folk medicinal uses of M. oleifera (MO) are attributed to the presence of functional bioactive compounds, such as phenolic acids, flavonoids, alkaloids, phytosterols, natural sugars, vitamins, minerals, and organic acids. The low molecular weight of M. oleifera cationic proteins (MOCP) extracted from the seeds is very useful and is used in water purification, because of its potent antimicrobial and coagulant properties. Also, the M. oleifera methyl esters (MOME) produced from the oil of the seeds meet the major specifications of the biodiesel standard of Germany, Europe, and United States (US). Thus, MO is emerging as one of the prominent industrial crops for sustainable biodiesel production in tropical and subtropical countries. In view of the high nutritional, nutraceutical, and industrial values, it is important to compile an updated comprehensive review on the related aspects of this multipurpose and miracle tree. Hence, the present study is focused on the nutritionally significant bioactives and medicinal and biological properties, to explore the potential applications of MO in nutritionally rich food preparations. Furthermore, water coagulation, proteins, and fatty acid methyl esters from the MO seeds are reviewed, to explore their possible industrial applications in biodiesel production and water purification. In addition, the future perspectives in these areas are suggested.