Cannabis Roots: Therapeutic, Biotechnological and Environmental Aspects.

Since the legalization of recreational cannabis in Canada in 2018, the number of licenses for this crop has increased significantly, resulting in an increase in waste generated. Nevertheless, cannabis roots were once used for their therapeutic properties, indicating that they could be valued today rather than dismissed. This review will focus on both traditional therapeutic aspects and potential use of roots in modern medicine while detailing the main studies on active phytomolecules found in cannabis roots. The environmental impact of cannabis cultivation and current knowledge of the root-associated microbiome are also presented as well as their potential applications in biotechnology and phytoremediation. Thus, several high added-value applications of cannabis roots resulting from scientific advances in recent years can be considered to remove them from discarded residues.

Thykamine Extracts from Spinach Reduce Acute Inflammation In Vivo and Downregulate Phlogogenic Functions of Human Blood Neutrophils In Vitro.

The anti-inflammatory and antioxidant role of Thykamine, a botanical extract of thylakoides obtained from spinach leaves, has been investigated in animal and cellular models. The oxidative properties have been proven by inhibiting NO production (>98%) in J774A.1 cells and by protecting a linoelic acid emulsion subjected to lipid peroxidation caused by AAPH. Thykamine injected intraperitoneally to rats reduced the inflammatory process of (TNBS)-induced colitis and carrageenan-induced paw edema. As neutrophils are the first cells to migrate to inflammatory sites, the influence of Thykamine on the primary neutrophil functions were studied. Thykamine dose-dependent reduced neutrophil chemiotaxis, phagocytosis, and degranulation. No change in the release of LDH by neutrophils on Thykamine was recorded. Thykamine inhibited by 85% the neutrophil production of O2-. A superoxide recovery activity was observed on a zymography demonstrating a SOD-like enzyme on Thykamine extracts. Spontaneous fluorescence provided by carotenoid and chlorophyll pigments (488/675 nm) detected Thykamine on the surface, in the cytoplasm (mainly central where Golgi are present) and weakly in the nucleus of neutrophils. The results argue that SOD and pigments found in Thykamine are part of its antioxidant and anti-inflammatory properties shown in in vivo and in vitro models of inflammation.

Comparison of topical antiaging creams in the management of lateral canthal lines.

OBJECTIVE: This study compared an antiaging treatment with two currently marketed cosmetic antiaging products for the treatment of lateral canthal lines ("crow's feet"). METHODS: Healthy female volunteers (72) aged of 54.6 years (mean) having fine-to-moderate wrinkles in the lateral canthal areas were randomized to one of three treatments applied daily over 28 days: Group A (Purgenesis™ Day Cream, Purgenesis™ Eye Cream, and Purgenesis™ Night Cream); Group B (Prevage® Eye Lotion, Prevage® Day Cream, and Prevage® Night Cream); or Group C (La Mer® Eye Balm, Crème de La Mer® , and La Mer® Night Cream). The effects on anti-wrinkle properties and for sensory attributes and general performance were evaluated on Days 1, 7, and 28. RESULTS: Skin hydration improved significantly at all time points in Groups A and B, and at Day 28 in Group C. Group A patients experienced significant improvements in measured skin elasticity parameters at Day 28; extensibility and maximum amplitude were significantly better at Day 28 in Groups B and C. Benefits were also seen in profilometric parameters with statistical significance only in Group A Volunteer tolerance was good with all three treatments, although moderate and high levels of adverse events were numerically higher in Group B than in Groups A or C, and levels of slight discomfort were significantly more prevalent in Group B. CONCLUSION: The Purgenesis™ antiaging treatment significantly improved skin hydration, elasticity, and profilometry parameters during a 28-day study. This therapy was found to be well tolerated and effective in countering the cutaneous signs of aging.

Study of antioxidant properties of thylakoids and application in UV protection and repair of UV-induced damage.

BACKGROUND: Skin is affected by environmental stress such as ultraviolet exposure. Topically applied antioxidants confer protection against this stress. Spinach thylakoid extracts are plant samples known as photosynthetic membranes containing antioxidant molecules able to dissipate excess of energy and oxidative stress. METHODS: Antioxidant contents and activities were tested in thylakoid extracts stored for different periods at 4°C to compare their efficacities. Cytotoxicity of thylakoids was tested on human THP-1 cells along with the capacity to protect from oxidative stress using flow cytometry. Protection of thylakoids against ultraviolet was tested on engineered human skin using two formulations and evaluated by electronic microscopy. RESULTS: Results indicate that thylakoid extracts possess antioxidant molecules that were not significantly affected by storage at 4°C whereas photosynthetic activity was storage-dependent. Thylakoid extracts were not cytotoxic to human THP-1 cells, and three extracts protected cells against reactive oxygen species. Moreover, formulation comprising 0.1% or 0.01% of thylakoids and sunscreen provided a synergetic protection against UV exposure. Thylakoid extracts mixed with a neutral cream were also able to repair UV damages on engineered human skin. CONCLUSIONS: Thylakoid extracts contained various antioxidant molecules, and their properties were maintained in over storage at 4°C for more than 72 months. Molecules and enzymes present in thylakoid extracts are involved in protecting and restoring the harmful effects of UV exposure. The involvement of antioxidant molecules such as carotenoids, SOD, and Fe-S clusters in cellular and regulatory metabolic reactions may explain the observed protective effects.

Application of Distributive Conjugal DNA Transfer in Mycobacterium smegmatis To Establish a Genome-Wide Synthetic Genetic Array.

Genetic redundancy can obscure phenotypic effects of single-gene mutations. Two individual mutations may be viable separately but are lethal when combined, thus synthetically linking the two gene products in an essential process. Synthetic genetic arrays (SGAs), in which defined mutations are combined, provide a powerful approach to identify novel genetic interactions and redundant pathways. A genome-scale SGA can offer an initial assignment of function to hypothetical genes by uncovering interactions with known genes or pathways. Here, we take advantage of the chromosomal conjugation system of Mycobacterium smegmatis to combine individual donor and recipient mutations on a genome-wide scale. We demonstrated the feasibility of a high-throughput mycobacterial SGA (mSGA) screen by using mutants of esx3, fxbA, and recA as query genes, which were combined with an arrayed library of transposon mutants by conjugation. The mSGA identified interacting genes that we had predicted and, most importantly, identified novel interacting genes-encoding both proteins and a noncoding RNA (ncRNA). In combination with other molecular genetic approaches, the mSGA has great potential to both reduce the high number of conserved hypothetical protein annotations in mycobacterial genomes and further define mycobacterial pathways and gene interactions.IMPORTANCE Mycobacterium smegmatis is the model organism of choice for the study of mycobacterial pathogens, because it is a fast-growing nonpathogenic species harboring many genes that are conserved throughout mycobacteria. In this work, we describe a synthetic genetic array (mSGA) approach for M. smegmatis, which combines mutations on a genome-wide scale with high efficiency. Analysis of the double mutant strains enables the identification of interacting genes and pathways that are normally hidden by redundant biological pathways. The mSGA is a powerful genetic tool that enables functions to be assigned to the many conserved hypothetical genes found in all mycobacterial species.

Intercellular communication and conjugation are mediated by ESX secretion systems in mycobacteria.

Communal bacterial processes require intercellular communication mediated by secretion systems to coordinate appropriate molecular responses. Intercellular communication has not been described previously in mycobacteria. Here we show that the ESX secretion-system family member ESX-4 is essential for conjugal recipient activity in Mycobacterium smegmatis Transcription of esx4 genes in the recipient requires coculture with a donor strain and a functional ESX-1 apparatus in the recipient. Conversely, mutation of the donor ESX-1 apparatus amplifies the esx4 transcriptional response in the recipient. The effect of ESX-1 on esx4 transcription correlates with conjugal DNA transfer efficiencies. Our data show that intercellular communication via ESX-1 controls the expression of its evolutionary progenitor, ESX-4, to promote conjugation between mycobacteria.

Substrate adaptabilities of Thermotogae mannan binding proteins as a function of their evolutionary histories.

The Thermotogae possess a large number of ATP-binding cassette (ABC) transporters, including two mannan binding proteins, ManD and CelE (previously called ManE). We show that a gene encoding an ancestor of these was acquired by the Thermotogae from the archaea followed by gene duplication. To address the functional evolution of these proteins as a consequence of their evolutionary histories, we measured the binding affinities of ManD and CelE orthologs from representative Thermotogae. Both proteins bind cellobiose, cellotriose, cellotetraose, ß-1,4-mannotriose, and ß-1,4-mannotetraose. The CelE orthologs additionally bind ß-1,4-mannobiose, laminaribiose, laminaritriose and sophorose while the ManD orthologs additionally only weakly bind ß-1,4-mannobiose. The CelE orthologs have higher unfolding temperatures than the ManD orthologs. An examination of codon sites under positive selection revealed that many of these encode residues located near or in the binding site, suggesting that the proteins experienced selective pressures in regions that might have changed their functions. The gene arrangement, phylogeny, binding properties, and putative regulatory networks suggest that the ancestral mannan binding protein was a CelE ortholog which gave rise to the ManD orthologs. This study provides a window on how one class of proteins adapted to new functions and temperatures to fit the physiologies of their new hosts.

Novel inositol catabolic pathway in Thermotoga maritima.

myo-inositol (MI) is a key sugar alcohol component of various metabolites, e.g. phosphatidylinositol-based phospholipids that are abundant in animal and plant cells. The seven-step pathway of MI degradation was previously characterized in various soil bacteria including Bacillus subtilis. Through a combination of bioinformatics and experimental techniques we identified a novel variant of the MI catabolic pathway in the marine hyperthermophilic bacterium Thermotoga maritima. By using in vitro biochemical assays with purified recombinant proteins we characterized four inositol catabolic enzymes encoded in the TM0412-TM0416 chromosomal gene cluster. The novel catabolic pathway in T. maritima starts as the conventional route using the myo-inositol dehydrogenase IolG followed by three novel reactions. The first 2-keto-myo-inositol intermediate is oxidized by another, previously unknown NAD-dependent dehydrogenase TM0412 (named IolM), and a yet unidentified product of this reaction is further hydrolysed by TM0413 (IolN) to form 5-keto-l-gluconate. The fourth step involves epimerization of 5-keto-l-gluconate to d-tagaturonate by TM0416 (IolO). T. maritima is unable to grow on myo-inositol as a single carbon source. The determined in vitro specificity of the InoEFGK (TM0418-TM0421) transporter to myo-inositol-phosphate suggests that the novel pathway in Thermotoga utilizes a phosphorylated derivative of inositol.

Yeast response to LA virus indicates coadapted global gene expression during mycoviral infection.

Viruses that infect fungi have a ubiquitous distribution and play an important role in structuring fungal communities. Most of these viruses have an unusual life history in that they are propagated exclusively via asexual reproduction or fission of fungal cells. This asexual mode of transmission intimately ties viral reproductive success to that of its fungal host and should select for viruses that have minimal deleterious impact on the fitness of their hosts. Accordingly, viral infections of fungi frequently do not measurably impact fungal growth, and in some instances, increase the fitness of the fungal host. Here we determine the impact of the loss of coinfection by LA virus and the virus-like particle M1 upon global gene expression of the fungal host Saccharomyces cerevisiae and provide evidence supporting the idea that coevolution has selected for viral infection minimally impacting host gene expression.

Use of chlorophyll a fluorescence to detect the effect of microcystins on photosynthesis and photosystem II energy fluxes of green algae.

The phenomenon of cyanobacteria bloom occurs widely in lakes, reservoirs, ponds and slow flowing rivers. Those blooms can have important repercussions, at once on recreational and commercial activities but also on the health of animals and human beings. Indeed, many species are known to produce toxins which are released in water mainly at cellular death. The cyanotoxin most frequently encountered is the microcystin (MC), a hepatotoxin which counts more than 70 variants. The use of fast tests for the detection of this toxin is thus a necessity for the protection of the ecosystems and the human health. A promising method for their detection is a bioassay based on the chlorophyll a fluorescence of algae. Many studies have shown that algae are sensible to diverse pollutants, but were almost never used for cyanotoxins. Therefore, our goals were to evaluate the effect of microcystin on the fluorescence of different species of algae and how it can affect the flow of energy through photosystem II. To reach these objectives, we exposed four green algae (Scenedesmus obliquus CPCC5, Chlamydomonas reinhardtii CC125, Pseudokirchneriella subcapitata CPCC37 and Chlorella vulgaris CPCC111) to microcystin standards (variants MC-LF, LR, RR, YR) and to microcystin extracted from Microcystis aeruginosa (CPCC299), which is known to produce mainly MC-LR. Chlorophyll a fluorescence was measured by PEA (Plant Efficiency Analyzer) and LuminoTox. The results of our experiment showed that microcystins affect the photosynthetic efficiency and the flow of energy through photosystem II from 0.01 µg/mL, within only 15 min. From exposure to standard of microcystin, we showed that MC-LF was the most potent variant, followed by MC-YR, LR and RR. Moreover, green algae used in this study demonstrated different sensitivity to MCs, S. obliquus being the more sensitive. We finally demonstrated that LuminoTox was more sensitive to MCs than parameters measured with PEA, although the latter brings indication on the mode of action of MCs at the photosynthetic apparatus level. This is the first report showing a photosynthetic response within 15 min of exposure. Our results suggest that bioassay based on chlorophyll fluorescence can be used as a rapid and sensitive tool to detect microcystin.

Ligands of thermophilic ABC transporters encoded in a newly sequenced genomic region of Thermotoga maritima MSB8 screened by differential scanning fluorimetry.

The chromosome of Thermotoga maritima strain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3' end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5' end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 µM, 0.300 µM, and 56.78 µM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound with K(d) (dissociation constant) values of 0.042 µM, 0.059 µM, and 1.436 µM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars: T. maritima MSB8 genomovar TIGR and T. maritima MSB8 genomovar DSM 3109, respectively.

Single-cell transcript analysis of human embryonic stem cells.

We demonstrate the qualitative and quantitative power of single-cell transcript analysis to characterize transcriptome dynamics in human embryonic stem cells (hESC's). Single-cell analysis can systematically determine unique cellular profiles for use in cell sorting and identification, show the potential to augment standing models of cellular differentiation, and elucidate the behavior of stem cells exiting pluripotency. Using single-cell analysis of H9 hESC's differentiating under three culture conditions, we revealed transient expression of mesendodermal markers in all three protocols, followed by increasingly stable expression of embryonic endoderm and extra-embryonic endoderm markers. Our single-cell profiles reveal mixed populations of cell types, with both transcriptional and temporal heterogeneity marking differentiation under all conditions. Interestingly, we also observe extensive and prolonged co-expression of markers regulating both pluripotency and lineage differentiation in all culture conditions, and we find that pluripotency marker transcripts remain detectable in the majority of cells for many days. Finally, we show that cells derived from undifferentiated hESC colonies display consistent gene expression profiles characterized by three cohorts of transcripts: uniform, absent and sporadically detected messages, and that a striking correlation exists between genes' membership in these cohorts and their hESC promoter chromatin state, with bivalent promoters dominating the sporadic transcripts.

Bioluminescent imaging of Trypanosoma brucei shows preferential testis dissemination which may hamper drug efficacy in sleeping sickness.

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs.

Mirtazapine and paroxetine in major depression: a comparison of monotherapy versus their combination from treatment initiation.

This double-blind study compared initial combination therapy against monotherapy using two antidepressant drugs with complementary mechanisms of action on the serotonin (5-HT) and norepinephrine (NE) systems. Sixty one adult patients with a DSM-IV diagnosis of unipolar depression were randomized to receive mirtazapine (30 mg/day), paroxetine (20 mg/day), or the combination of both drugs for 6 weeks. Response at week 4 was defined as a 30% reduction in the Montgomery-Asberg Depression Rating Scale (MADRS), and at week 6 as a 50% reduction in the MADRS. Remission was defined as a reduction in the MADRS score to 10 points or less. After 4 weeks, non-responders in the monotherapy groups had their medication dose increased by 50%. After 6 weeks, non-responders on monotherapy had the second trial drug added to their current regimen. Non-responders on combination therapy had the dosage of both drugs increased by 50%. There was a significantly greater decrease in MADRS scores in the combination group compared to the monotherapy groups at days 28, 35 and 42, with a 10 point difference separating the combination from the monotherapies at day 42. Remission rates at week 6 were 19% on mirtazapine, 26% on paroxetine, and 43% on the combination. Fifteen patients in the mirtazapine arm and 10 in the paroxetine arm who did not respond had the other drug added to their current regimen, and 5 on the combination had an increase in dose of both drugs secondary to non-response. Of these 30 patients, approximately 50% went on to achieve remission in the subsequent 2 weeks. These results indicate that the combined use of two antidepressants was well tolerated and produced a greater improvement than monotherapy.

The short chain fatty acid butyrate induces promoter demethylation and reactivation of RARbeta2 in colon cancer cells.

It has been proposed that cancer prevention results from multiple dietary agents acting together as "action packages." Here we obtain evidence that butyrate, which is generated from dietary fiber, enhances the responsiveness of colon cancer cells to all-trans retinoic acid (ATRA). Evidence was obtained that this interaction depends on histone deactylase one (HDAC1) inhibition by butyrate and retinoic acid receptor alpha (RARalpha) activation by ATRA. The enhancement of RAR beta 2 (RARbeta2) activation was accompanied by a rapid demethylation of the RARbeta2 promoter. This demethylation could be achieved by butyrate alone, and it differed from that triggered by the DNA methyltransferase inhibitor 5-Aza-2' deoxycytidine in that it was 1) sporadic on the RARbeta2 promoter, 2) not genome wide, and 3) independent of extensive DNA replication. An analysis of inter-methylated sites assay indicated that only a few percent of loci analyzed showed reduced methylation. In colon cancer cells that were particularly resistant to RARbeta2 reactivation, the actions of butyrate could be further enhanced by the soy isoflavone genistein, which has also been reported to work through an epigenetic mechanism. These data suggest that dietary compounds that modulate epigenetic programming are likely to function best in the presence of retinoids and other cancer-preventing compounds that are sensitive to a cell's epigenetic state.

An essential cell cycle-regulated nucleolar protein relocates to the mitotic spindle where it is involved in mitotic progression in Trypanosoma brucei.

TbNOP86 and TbNOP66 are two novel nucleolar proteins isolated in Trypanosoma brucei. They share 92.6% identity, except for an additional C-terminal domain of TbNOP86 of 182 amino acids in length. Both proteins are found in Trypanosomatidae, but similarity to other eukaryotic proteins could not be found. TbNOP86 and TbNOP66 are expressed at similar level in procyclic and bloodstream forms, although the relative level of expression of TbNOP66 is 11 times lower. TbNOP86 undergoes post-translational modifications, as it is found predominantly at 110 kDa compared with the predicted 86 kDa. Immunofluorescence of overexpressed ty-tagged TbNOP86 and TbNOP66 showed that both proteins accumulated in the nucleolus of G(1) cells. This was confirmed by the co-localization of an endogenous TbNOP86-myc with the nucleolar protein Nopp140. TbNOP86-ty localization is cell cycle-regulated, because it colocalizes with the mitotic spindle in mitotic cells. TbNOP86 is required for mitotic progression in both life stages as depleted cells are enriched in the G(2)/M phase. In procyclic cells, a reduced growth rate is accompanied by an accumulation of zoids (0N1K), 2N1K, and multinucleated cells (xNyK). The 2N1K cells are blocked in late mitosis as nucleolar segregation is completed. TbNOP86 depletion in bloodstream form caused a drastic growth inhibition producing cells bearing two kinetoplasts and an enlarged nucleus (1N(*)2K), followed by an accumulation of 2N2K cells with connected nuclei and xNyK cells. These studies of TbNOP86 provide a more comprehensive account of proteins involved in mitotic events in trypanosomes and should lead to the identification of partners with similar function.

Combined use of photosynthetic enzyme complexes and microalgal photosynthetic systems for rapid screening of wastewater toxicity.

Because of the often episodic nature of wastewater toxicity, routine monitoring using expensive and time consuming tests can constitute an inefficient means of toxicity evaluation, particularly when negative results are generated. Cost-effective screening tests enabling the rapid detection of effluent toxicity are clearly needed, and they should be used to rapidly determine where in-depth investigations should be focused. The LuminoTox is a recently-developed screening test enabling the rapid determination of wastewater toxicity. This test is based on the inhibition of chlorophyll fluorescence emitted by photosynthetic systems. The combined use of photosynthetic enzyme complexes (PECs), isolated from higher plants, and whole photosynthetic organisms (algae) allows a wide range of toxic inhibitors to be detected within 10-15 min. The detection thresholds obtained for individual toxic chemicals indicate that algae are less sensitive to metal cations than PECs, because of the algal cell wall being ion selective. However, other toxic chemicals, such as phenolic compounds and nitrogen ammonia, acting on the last constituents of the photosynthetic enzyme complex that are degraded during the PEC extraction process, are more easily detected with algae after just 10 min of exposure. The combination of PECs and algae is not only useful for rapid toxicity screening, but yields results that are as sensitive as those of standard bioassays. Toxicity data generated with mining industry effluents demonstrate that PECs routinely prove to be as sensitive as daphnia, while algal sensitivity is comparable to that of the standard trout bioassay. An important feature of LuminoTox and algal photosynthetic system testing, however, resides in the production of their rapid and sensitive responses (10-15 min) in comparison with those of the more traditional tests (48-96 h for daphnia and trout, respectively).

Identification of methicillin-resistant Staphylococcus aureus carriage in less than 1 hour during a hospital surveillance program.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide and is responsible for significant morbidity, mortality, and health care costs. Control strategies to limit the emergence and spread of this organism rely on rapid and sensitive tests for detection of MRSA carriage. However, the standard surveillance culture method for detecting MRSA is labor intensive and time-consuming (2-3 days per procedure). There is thus a need for a rapid and accurate method to screen for MRSA carriage. METHODS: We recently developed an easy-to-use real-time polymerase chain reaction (PCR) assay suitable for specific detection of MRSA in nasal specimens in <1 h. We studied the efficacy of our new PCR assay in routine screening for nasal MRSA carriage during a hospital surveillance program. A total of 331 nasal specimens obtained from 162 patients at risk for colonization were tested by both the standard mannitol agar culture method and our PCR assay. RESULTS: The PCR assay detected MRSA in all 81 samples that were culture positive for MRSA. The PCR assay detected 4 additional MRSA-positive specimens, for a specificity of 98.4%, a positive predictive value of 95.3%, and a sensitivity and negative predictive value of 100%. CONCLUSIONS: This novel PCR assay allows reliable identification of MRSA carriers in <1 h. This test should facilitate the efficacy of MRSA surveillance programs.

The ribosomal RNA gene promoter and adjacent cis-acting DNA sequences govern plasmid DNA partitioning and stable inheritance in the parasitic protozoan Leishmania.

Detailed analysis of the Leishmania donovani ribosomal RNA (rRNA) gene promoter region has allowed the identification of cis-acting sequences involved in plasmid DNA partitioning and stable plasmid inheritance. We report that plasmids bearing the 350 bp rRNA promoter along with the 200 bp region immediately 3' to the promoter exhibited a 6.5-fold increase in transformation frequency and were transmitted to daughter cells as single-copy molecules. This is in contrast to what has been observed for plasmid molecules in this organism so far. Moreover, we show that these low-copy-number plasmids displayed a remarkable mitotic stability in the absence of selective pressure. The region in the vicinity of the RNA pol I transcription initiation site, and also in the adjacent 200 nt, displays a complex structural organization and shares sequence similarity to the yeast autonomously replicating consensus sequence and centromere DNA elements. Deletion analyses indicated that these elements were necessary but not sufficient for plasmid DNA partitioning and stable inheritance, and that the rRNA promoter region was required for optimal function. These results suggest an interplay between RNA pol I transcription, DNA replication, DNA partitioning and mitotic stability in trypanosomatids. This is the first example of defined DNA elements for plasmid partitioning and stable inheritance in the protozoan parasite Leishmania.

Ala67Thr polymorphism in the Agouti-related peptide gene is associated with inherited leanness in humans.

A role for melanocortin signaling in the regulation of body weight in humans has been clearly established. Haploinsufficiency of the type 4 melanocortin receptor is associated with early-onset obesity, implying that this receptor provides an important tonic inhibition of weight gain. Agouti-related peptide (AGRP) is an endogenous antagonist of melanocortin signaling. Therefore, loss of AGRP function could lead to the expression of a lean phenotype. We investigated the potential role of AGRP in human weight regulation by examining the association between the Ala67Thr AGRP polymorphism and indices of body composition. Significant associations were found between homozygosity for this mutation (n = 8) and body composition phenotype in 874 subjects of the Quebec family study (QFS). By PCR-RFLP analysis, we have identified eight individuals who are homozygous for the 67Thr variant allele within the QFS population, where none were observed in SAFHS. The eight QFS homozygote individuals have lower weight (-16%; P = 0.02), body mass index (-17%; P = 0.01), fat free mass (-9%; P = 0.002), fat mass (FM) (-20%; P = 0.04), and leptin (-20%; P = 0.02) when compared to those carrying at least one 67Ala allele. Individuals homozygous for the 67Thr allele had a BMI that was either at or slightly below an ideal range for their age. Thus, the Ala67Thr AGRP polymorphism is associated with lower body weight in humans, with the largest effect being observed on body FM. We did not observe any difference in the stability or cellular distribution of the mutant protein in a heterologous expression system, thus the mechanism of this effect requires further investigation.