Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed ß-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

Mutations in the drug resistance-determining region of Mycobacterium lepromatosis isolated from leprosy patients in Mexico.

Mycobacterium lepromatosis, an independent species from Mycobacterium leprae, has been found to be a causative agent for diffuse lepromatous leprosy (DLL) in Mexico, but remains poorly studied. Here, the drug resistance-determining regions (DRDR) of folP1, rpoB and gyrA (conferring resistance to dapsone, rifampicin and quinolone, respectively) in M. lepromatosis from leprosy patients in Mexico were characterized. No mutations or silent mutations were found at previously characterized major sites in DRDR of M. lepromatosis. However, a non-synonymous mutation was found in codon 54 between two major sites of the folP1 DRDR in M. lepromatosis sequences. All M. lepromatosis isolates showed CAG sequence in codon 54 of folP1. Because the next codons 53 and 55 are known as major mutation sites for drug resistance, more detailed analysis using more samples is needed to determine whether it influences susceptibility to dapsone and/or efficiency of folate biosynthesis in M. lepromatosis or not.

Variations in IL-23 and IL-25 receptor gene structure, sequence and expression associated with the two disease forms of sheep paratuberculosis.

The immunopathology of paucibacillary and multibacillary sheep paratuberculosis is characterized by inflammatory T cell and macrophage responses respectively. IL-23 and IL-25 are key to the development of these responses by interaction with their complex receptors, IL-23R/IL-12RB1 and IL-17RA/IL-17RB. In humans, variations in structure, sequence and/or expression of these genes have been implicated in the different pathological forms of tuberculosis and leprosy, and in gastrointestinal inflammatory disorders such as Crohn's disease. Sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for all the identified variants and used to compare expression in the ileo-caecal lymph node of sheep with paucibacillary or multibacillary paratuberculosis and uninfected animals. With IL-23 receptor, only the IL12RB1v3 variant, which lacks the receptor activation motif was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Of the IL17RB variants only full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology; which may also contribute to Th2 polarization. IL17RA expression was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology.

AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

Yeast diversity in a traditional French cheese "Tomme d'orchies" reveals infrequent and frequent species with associated benefits.

This study is aimed at unrevealing the yeast diversity of handmade cheese, Tomme d'orchies, produced and marketed in the north of France. A total of 185 yeast colonies were isolated from the surface and core of this cheese. From these, 80 morphologically different colonies were selected and subjected to rep-PCR analysis. The isolates were clustered into six distinct groups based on their DNA fingerprints. From each group, at least 30% of isolates were selected and identified to species level by biochemical characteristics (ID32C Api system) and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA regions. The isolates belonged to Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus, frequently isolated, and less frequently isolated Saturnispora mendoncae and Clavispora lusitaniae. Two isolates designated as Kluyveromyces lactis (isolate S-3-05) and Kluyveromyces marxianus (isolate S-2-05) were non-hemolytic, sensitive to antifungal compounds and able to inhibit the growth of pathogens including Candida albicans, Listeria monocytogenes and some bacilli.

The Potential Use of Natural and Structural Analogues of Antimicrobial Peptides in the Fight against Neglected Tropical Diseases.

Recently, research into the development of new antimicrobial agents has been driven by the increase in resistance to traditional antibiotics and Emerging Infectious Diseases. Antimicrobial peptides (AMPs) are promising candidates as alternatives to current antibiotics in the treatment and prevention of microbial infections. AMPs are produced by all known living species, displaying direct antimicrobial killing activity and playing an important role in innate immunity. To date, more than 2000 AMPs have been discovered and many of these exhibit broad-spectrum antibacterial, antiviral and anti-parasitic activity. Neglected tropical diseases (NTDs) are caused by a variety of pathogens and are particularly wide-spread in low-income and developing regions of the world. Alternative, cost effective treatments are desperately needed to effectively battle these medically diverse diseases. AMPs have been shown to be effective against a variety of NTDs, including African trypanosomes, leishmaniosis and Chagas disease, trachoma and leprosy. In this review, the potential of selected AMPs to successfully treat a variety of NTD infections will be critically evaluated.

Occurrence of FFZ genes in yeasts and correlation with fructophilic behaviour.

Fructophily has been described in yeasts as the ability to utilize fructose preferentially when fructose and glucose are available in the environment. In Zygosaccharomyces bailii and Zygosaccharomyces rouxii, fructophilic behaviour has been associated with the presence of a particular type of high-capacity and low-affinity fructose transporters designated Ffz. In this study, a PCR screening was performed in several yeasts using degenerate primers suitable to detect FFZ-like genes. In parallel, fructophilic character was evaluated in the same strains by comparing the relative consumption rate of fructose and glucose. For all the strains in which FFZ-like genes were detected, fructophilic behaviour was observed (25 strains). Results show that FFZ genes are ubiquitous in the Zygosaccharomyces and Starmerella clades. Strains of Lachancea fermentati, Torulaspora microellipsoides and Zygotorulaspora florentina were not fructophilic and did not harbour FFZ genes. It is of note that these new species were recently removed by taxonomists from the Zygosaccharomyces clade, supporting the view that the presence of FFZ-like genes is a main characteristic of Zygosaccharomyces. Among the strains tested, only Hanseniaspora guilliermondii NCYC2380 was an exception, having a preference for fructose in medium with high sugar concentrations, despite no FFZ-like genes being detected in the screening. Furthermore, this study supports the previous idea of the emergence of a new family of hexose transporters (Ffz facilitators) distinct from the Sugar Porter family.

Hanseniaspora jakobsenii sp. nov., a yeast isolated from Bandji, a traditional palm wine of Borassus akeassii.

Investigation of the microbial diversity of Bandji, a traditional palm wine from Burkina Faso (West Africa) revealed the presence of two yeast isolates (YAV16 and YAV17T) with unusual phenotypic and genotypic characteristics. The isolates divide by bipolar budding with no production of ascospores. Phylogenetic analysis of concatenated sequences of the 26S rRNA gene D1/D2 and internal transcribed spacer (ITS) regions indicated that the novel species was most closely related to Kloeckera lindneri and Hanseniaspora valbyensis. The new isolates differed from K. lindneri NRRL Y-17531T and H. valbyensis CBS 479T by substitutions in the D1/D2 region of 12 and 16 nt respectively. The divergence in the ITS region from the closely related species was characterized by substitutions of 45-46 nt. Repetitive palindromic PCR (rep-PCR) profiles of YAV16 and YAV17T were also significantly different from those of K. lindneri MUCL 31146T ( = NRRL Y-17531T), H. valbyensis NCYC 17T ( = CBS 479T) and other species of the genus Hanseniaspora. Based on the results of the phenotypic and genotypic characterizations, it was concluded that the new isolates represent a novel species for which the name Hanseniaspora jakobsenii sp. nov. is proposed with YAV17T ( = CBS 12942T = DSM 26339T = NCYC 3828T; MycoBank number MB 805785) as the type strain.

Assessment of endophytic yeast diversity in rice leaves by a culture-independent approach.

Endophytic microorganisms inhabit internal plant tissues in the host plant without causing any symptoms or negative effects. Although the diversity of endophytes has been evaluated by both culture-dependent and culture-independent methods, less information is available on yeast communities. Therefore, in this study a culture-independent method was used to examine endophytic yeasts associated with rice leaves based on the large subunit of ribosomal DNA using a semi-nested PCR technique. Sequence analysis indicated that the colonization frequency and the relative species frequency (RF) of endophytic yeast phylotypes were 0.41 and 0.06, respectively, and the majority of the yeast phylotypes were basidiomycetous yeasts. The phylotypes were designated as five known species (Cryptococcus victoriae, Debaryomyces hansenii, Debaryomyces vindobonensis, Meyerozyma guilliermondii and Pseudozyma antarctica), together with seventeen phylotypes closest to Candida metapsilosis, Cryp. foliicola, Cryp. laurentii, Pseudozyma abaconensis, Pseudozyma aphidis and Trichosporon asahii, among which some could be novel species. The most prevalent phylotypes were those closest to Cryp. foliicola (47.5 % RF) followed by D. hansenii (22.8 % RF) and P. antarctica (16.8 % RF). The presence of the phylotypes related to species known for their potential applications as biocontrol agents and plant growth promoting hormone producers suggests that they may have valuable applications. In addition, our findings revealed the occurrence of novel phylotypes at high frequency, which should encourage extensive studies to discover novel yeast species and to understand their roles in the rice leaves.

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

UNLABELLED: Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. IMPORTANCE: RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain.

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens.

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes. CiLV-C and CiLV-N are present in South and Central America and as far north as parts of Mexico. Although leprosis disease was originally described from Florida, it disappeared from there in the 1960s. The United States Department of Agriculture-Agricultural Research Service maintains preserved citrus specimens identified at inspection stations 50 or more years ago with symptoms of citrus leprosis. We isolated RNA from these samples and performed degradome sequencing. We obtained nearly full-length genome sequences of both a typical CiLV-C isolate intercepted from Argentina in 1967 and a distinct CiLV-N isolate obtained in Florida in 1948. The latter is a novel form of CiLV-N, not known to exist anywhere in the world today. We have also documented the previously unreported presence of CiLV-N in Mexico in the mid-20th century.

Insight into the evolution and origin of leprosy bacilli from the genome sequence of Mycobacterium lepromatosis.

Mycobacterium lepromatosis is an uncultured human pathogen associated with diffuse lepromatous leprosy and a reactional state known as Lucio's phenomenon. By using deep sequencing with and without DNA enrichment, we obtained the near-complete genome sequence of M. lepromatosis present in a skin biopsy from a Mexican patient, and compared it with that of Mycobacterium leprae, which has undergone extensive reductive evolution. The genomes display extensive synteny and are similar in size (∼3.27 Mb). Protein-coding genes share 93% nucleotide sequence identity, whereas pseudogenes are only 82% identical. The events that led to pseudogenization of 50% of the genome likely occurred before divergence from their most recent common ancestor (MRCA), and both M. lepromatosis and M. leprae have since accumulated new pseudogenes or acquired specific deletions. Functional comparisons suggest that M. lepromatosis has lost several enzymes required for amino acid synthesis whereas M. leprae has a defective heme pathway. M. lepromatosis has retained all functions required to infect the Schwann cells of the peripheral nervous system and therefore may also be neuropathogenic. A phylogeographic survey of 227 leprosy biopsies by differential PCR revealed that 221 contained M. leprae whereas only six, all from Mexico, harbored M. lepromatosis. Phylogenetic comparisons indicate that M. lepromatosis is closer than M. leprae to the MRCA, and a Bayesian dating analysis suggests that they diverged from their MRCA approximately 13.9 Mya. Thus, despite their ancient separation, the two leprosy bacilli are remarkably conserved and still cause similar pathologic conditions.

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: a study from Eastern India.

BACKGROUND: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method. AIM: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. METHODS: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. RESULTS: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). LIMITATIONS: The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. CONCLUSION: The study indicates the existence of rifampicin drug resistance in Eastern India.

Diversity of yeast and mold species from a variety of cheese types.

To generate a comprehensive profile of viable fungi (yeasts and molds) on cheese as it is purchased by consumers, 44 types of cheese were obtained from a local grocery store from 1 to 4 times each (depending on availability) and sampled. Pure cultures were obtained and identified by DNA sequence of the ITS region, as well as growth characteristics and colony morphology. The yeast Debaryomyces hansenii was the most abundant fungus, present in 79 % of all cheeses and 63 % of all samples. Penicillium roqueforti was the most common mold, isolated from a variety of cheeses in addition to the blue cheeses. Eighteen other fungal species were isolated, ten from only one sample each. Most fungi isolated have been documented from dairy products; a few raise potential food safety concerns (i.e. Aspergillus flavus, isolated from a single sample and capable of producing aflatoxins; and Candida parapsilosis, an emerging human pathogen isolated from three cheeses). With the exception of D. hansenii (present in most cheese) and P. roqueforti (a necessary component of blue cheese), no strong correlation was observed between cheese type, manufacturer, or sampling time with the yeast or mold species composition.

The thalidomide-binding domain of cereblon defines the CULT domain family and is a new member of the ß-tent fold.

Despite having caused one of the greatest medical catastrophies of the last century through its teratogenic side-effects, thalidomide continues to be an important agent in the treatment of leprosy and cancer. The protein cereblon, which forms an E3 ubiquitin ligase compex together with damaged DNA-binding protein 1 (DDB1) and cullin 4A, has been recently indentified as a primary target of thalidomide and its C-terminal part as responsible for binding thalidomide within a domain carrying several invariant cysteine and tryptophan residues. This domain, which we name CULT (cereblon domain of unknown activity, binding cellular ligands and thalidomide), is also found in a family of secreted proteins from animals and in a family of bacterial proteins occurring primarily in δ-proteobacteria. Its nearest relatives are yippee, a highly conserved eukaryotic protein of unknown function, and Mis18, a protein involved in the priming of centromeres for recruitment of CENP-A. Searches for distant homologs point to an evolutionary relationship of CULT, yippee, and Mis18 to proteins sharing a common fold, which consists of two four-stranded ß-meanders packing at a roughly right angle and coordinating a zinc ion at their apex. A ß-hairpin inserted into the first ß-meander extends across the bottom of the structure towards the C-terminal edge of the second ß-meander, with which it forms a cradle-shaped binding site that is topologically conserved in all members of this fold. We name this the ß-tent fold for the striking arrangement of its constituent ß-sheets. The fold has internal pseudosymmetry, raising the possibility that it arose by duplication of a subdomain-sized fragment.

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

Association of a mutation in LACC1 with a monogenic form of systemic juvenile idiopathic arthritis.

OBJECTIVE: The pathologic basis of systemic juvenile idiopathic arthritis (JIA) is a subject of some controversy, with evidence for both autoimmune and autoinflammatory etiologies. Several monogenic autoinflammatory disorders have been described, but thus far, systemic JIA has only been attributed to a mutation of MEFV in rare cases and has been weakly associated with the HLA class II locus. This study was undertaken to identify the cause of an autosomal-recessive form of systemic JIA. METHODS: We studied 13 patients with systemic JIA from 5 consanguineous families, all from the southern region of Saudi Arabia. We used linkage analysis, homozygosity mapping, and whole-exome sequencing to identify the disease-associated gene and mutation. RESULTS: Linkage analysis localized systemic JIA to a region on chromosome 13 with a maximum logarithm of odds score of 11.33, representing the strongest linkage identified to date for this disorder. Homozygosity mapping reduced the critical interval to a 1.02-Mb region defined proximally by rs9533338 and distally by rs9595049. Whole-exome sequencing identified a homoallelic missense mutation in LACC1, which encodes the enzyme laccase (multicopper oxidoreductase) domain-containing 1. The mutation was confirmed by Sanger sequencing and segregated with disease in all 5 families based on an autosomal-recessive pattern of inheritance and complete penetrance. CONCLUSION: Our findings provide strong genetic evidence of an association of a mutation in LACC1 with systemic JIA in the families studied. Association of LACC1 with Crohn's disease and leprosy has been reported and justifies investigation of its role in autoinflammatory disorders.

A report of rifampin-resistant leprosy from northern and eastern India: identification and in silico analysis of molecular interactions.

Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae (M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy.

The vintage effect overcomes the terroir effect: a three year survey on the wine yeast biodiversity in Franciacorta and Oltrepò Pavese, two northern Italian vine-growing areas.

A three year survey on the dominant yeast populations in samples of air, must and wine in different vineyards and cellars of two northern Italian vine-growing territories (six sites in Franciacorta and eight sites in Oltrepò Pavese areas) was carried out. A total of 505 isolates were ascribed to 31 different species by RFLP analysis of the ITS1-5.8SrRNA-ITS2 region and partial sequence analysis of the 26S rRNA gene. The most commonly found species were Saccharomyces cerevisiae (frequency, F' = 58.7%; incidence, I' = 53.5%), Hanseniaspora uvarum (F' = 14.3%; I' = 5.3%), Metschnikowia fructicola (F' = 11.1%; I' = 5.0%) and Torulaspora delbrueckii (F' = 10.3%; I' = 3.8%). Among 270 S. cerevisiae new isolates, 156 (57.8%) revealed a different genetic pattern through polymorphism analysis of the interdelta regions by capillary electrophoresis, while 47 isolates (17.4 %) were clones of starter cultures. By considering the Shannon-Wiener index and results of principal component analysis (PCA) analyses, the year of isolation (vintage) proved to be a factor that significantly affected the biodiversity of the yeast species, whereas the geographical site (terroir) was not. Seventy-five per cent of S. cerevisiae isolates gathered in a unique cluster at a similarity level of 82%, while the remaining 25% were separated into minor groups without any evident relationship between δ-PCR profile and territory, year or source of isolation. However, in six cases a similar strain appeared at the harvesting time both in Franciacorta and Oltrepò Pavese areas, whereas surprisingly no strain was reisolated in the same vineyard or cellar for consecutive years.