Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa

Characterization of optimal CTL epitopes in Gag can provide crucial information for evaluation of candidate vaccines in populations at the epicenter of the HIV-1 epidemic. We screened 38 individuals with recent subtype C HIV-1 infection using overlapping consensus C Gag peptides and hypothesized that unique HLA-restricting alleles in the southern African population would determine novel epitope identity. Seventy-four percent of individuals recognized at least one Gag peptide pool. Ten epitopic regions were identified across p17, p24, and p2p7p1p6, and greater than two-thirds of targeted regions were directed at: TGTEELRSLYNTVATLY (p17, 35%); GPKEPFRDYVDRFFKTLRAEQATQDV (p24, 19%); and RGGKLDKWEKIRLRPGGKKHYMLKHL (p17, 15%). After alignment of these epitopic regions with consensus M and a consensus subtype C sequence from the cohort, it was evident that the regions targeted were highly conserved. Fine epitope mapping revealed that five of nine identified optimal Gag epitopes were novel: HLVWASREL, LVWASRELERF, LYNTVATLY, PFRDYVDRFF, and TLRAEQATQD, and were restricted by unique HLA-Cw*08, HLA-A*30/B*57, HLA-A*29/B*44, and HLA-Cw*03 alleles, respectively. Notably, three of the mapped epitopes were restricted by more than one HLA allele. Although these epitopes were novel and restricted by unique HLA, they overlapped or were embedded within previously described CTL epitopes from subtype B HIV-1 infection. These data emphasize the promiscuous nature of epitope binding and support our hypothesis that HLA diversity between populations can shape fine epitope identity, but may not represent a constraint for universal recognition of Gag in highly conserved domains.     

Identification by fluorescence spectroscopy of lactic acid bacteria isolated from a small-scale facility producing traditional dry sausages

Three different fluorescence spectra were recorded following excitation at 250 nm (aromatic amino acids+nucleic acids, AAA+NA), 316 nm (NADH) and 380 nm (FAD) for 20 type strain collections of lactic acid bacteria (LAB). Evaluation of the data using principal component analysis and factorial discriminant analysis showed a good discrimination of considered LAB at the genus, species and genus-species level. AAA+NA fluorophores showed the highest percentage of good classification. From AAA+NA spectra recorded on LAB isolated from a small-scale facility producing traditional dry sausages, we succeeded to identify 28 of 29 wild strains. This method allowed us to discriminate between Lactobacillus sakei subsp. carnosus and Lactobacillus sakei subsp. sakei. Thus, intrinsic fluorescence is an economical and powerful tool for the identification of wild LAB isolated from meat and meat products.     

Ultrastructure of the abdominal sense organ of the scallop <Emphasis Type="Italic">Mizuchopecten yessoensis</Emphasis> (Jay)

The sensory epithelium of the abdominal sense organ (ASO) of the scallop Mizuchopecten yessoensis is composed of three cell types, sensory cells, mucous cells, and multiciliated cells. Sensory cells bear a single long (up to 250 microm) cilium surrounded by an inner ring of nine modified microvilli and an outer ring of ordinary microvilli paired with modified microvilli. Sensory cells make up about 90% of the total number of cells in the sensory epithelium. Mucous cells, which are much wider than sensory cells, bear only ordinary microvilli on their apical surface. Rare multiciliated cells with short (4-6 microm) cilia are scattered in the periphery of the sensory epithelium sheet. All hairs, cilium, and microvilli of each sensory cell are interconnected by a fibrous network. Nine modified microvilli of a single cell are interconnected by prominent laterally running fibrous links. Membrane-associated electron-dense material of modified microvilli is connected to the ciliary membrane-associated electron-dense material by fine string-like links. These links mechanically bridge the space between the cilium and modified microvilli, as do mechanical links, described for the stereocilia and kinocilium of vertebrate vestibular and cochlear hair cells. The proximal portion of a sensory cilium is about 100 microm long and has a typical 9 x 2+2 axoneme arrangement. The distal portion of a cilium is approximately 2 times thinner than the proximal one and is filled with homogeneous electron-dense material. Along the distal portion, diffuse material associated with the external surface of the membrane is found. The rigidity of distal portion of a cilium is much less than that of the proximal one.     

Development of a system for integrative and stable transformation of the zygomycete<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis> by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated DNA transfer

Two transformation systems, based on the use of CaCl₂/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS⁺ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl₂/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl₂/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at Communicated by C. P. Hollenberg     

Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85

Mycolic acids, long-chain (C70-C90) alpha-alkyl, beta-hydroxy fatty acids, are characteristic cell envelope components of mycobacteria; similar but shorter-chain substances occur in corynebacteria and related taxa. These compounds apparently play an important role in the physiology of these bacteria. The deduced N-terminal region of PS1, one of the two major secreted proteins of Corynebacterium glutamicum encoded by the csp1 gene, is similar to the antigens 85 complex of Mycobacterium tuberculosis which has been shown to be associated in vitro with a mycoloyltransferase activity onto trehalose. Overexpression of PS1 in the wild-type strain of C. glutamicum suggested the implication of the protein in the transfer of corynomycolates, evidenced by an increase esterification of the cell wall arabinogalactan with corynomycolic acid residues and an accumulation of trehalose dicorynomycolates. Overexpression of truncated forms of PS1 demonstrated that the crucial region for transfer activity of the protein involves all the region of homology with antigens 85. To establish the putative mycoloyltransferase activity of PS1, a csp1-inactivated mutant of C. glutamicum was biochemically characterized. Inactivation of the gene resulted in: (i) a 50% decrease in the cell wall corynomycolate content; (ii) the alteration of the permeability of the C. glutamicum cell envelope; (iii) the decrease of the trehalose dicorynomycolate content; (iv) the accumulation of trehalose monocorynomycolate; and (v) the appearance of a glycolipid identified as 6-corynomycoloylglucose. Complementation of the mutant by the csp1 gene fully restored the wild-type phenotype. Finally, a mycoloyltransferase assay established that PS1 possesses a trehalose mycoloyltransferase activity. To define the in vivo function of antigens 85, the csp1-inactivated mutant was complemented with the fbpA, fbpB or fbpC genes. Complementation with the different fbp genes restored the normal cell wall corynomycolate content and permeability, but did not affect either the fate of trehalose corynomycolates or the occurrence of glucose corynomycolate. Thus, PS1 is one of the enzymes that transfer corynomycoloyl residues onto both the cell wall arabinogalactan and trehalose monocorynomycolate, whereas in the whole bacterium the mycobacterial antigens 85A, 85B and 85C can transfer mycolates only onto the cell wall acceptor in C. glutamicum.     

Camptotheca acuminata 10‐hydroxycamptothecin O‐methyltransferase: an alkaloid biosynthetic enzyme co‐opted from flavonoid metabolism

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10‐hydroxycamptothecin, and 10‐methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10‐hydroxycamptothecin O‐methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A‐ring 7‐OH of flavonoids, which is structurally equivalent to the 10‐OH of 10‐hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3‐D positioning of the 7‐OH, A‐ and C‐rings of flavonoids is nearly identical to the 10‐OH, A‐ and B‐rings of 10‐hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10‐hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7‐OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10‐hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non‐inhibitory flavonoid glycosides.     

Prevalence of sleep disorders and their impact on academic performance in medical students/University of Duhok

Full-time students experiencing high levels of stress due to a high bulk of teaching materials and academic performance demands are the most susceptible population class for different types of sleep disorders. The current study examined the prevalence of sleep disorders and their impacts on academic performance of a random sample of medical college students. In this regard, a random sample of 316 medical students of a large public university in Iraq participated in a cross-sectional study. The participants completed the SLEEP-50 self-reported questionnaire and questions about socio-demographic factors. The variables set included sleep apnea, insomnia, narcolepsy, restless legs syndrome, circadian rhythm sleep disorder, sleepwalking, nightmares, grade point average, and some socio-demographic characteristics. The study showed that to some extent, the students suffer from different types of sleep disorders with no substantial difference between males and females. Students with worse level of sleep disorders had a lower grade point average compared with those with normal sleep patterns (p = 0.001). The study confirmed that students with sleep disorders had poorer academic performance at college.

     

Structural basis for specific inhibition of the highly sensitive ShHTL7 receptor

Striga hermonthica is a root parasitic plant that infests cereals, decimating yields, particularly in sub‐Saharan Africa. For germination, Striga seeds require host‐released strigolactones that are perceived by the family of HYPOSENSITIVE to LIGHT (ShHTL) receptors. Inhibiting seed germination would thus be a promising approach for combating Striga. However, there are currently no strigolactone antagonists that specifically block ShHTLs and do not bind to DWARF14, the homologous strigolactone receptor of the host. Here, we show that the octyl phenol ethoxylate Triton X‐100 inhibits S. hermonthica seed germination without affecting host plants. High‐resolution X‐ray structures reveal that Triton X‐100 specifically plugs the catalytic pocket of ShHTL7. ShHTL7‐specific inhibition by Triton X‐100 demonstrates the dominant role of this particular ShHTL receptor for Striga germination. Our structural analysis provides a rationale for the broad specificity and high sensitivity of ShHTL7, and reveals that strigolactones trigger structural changes in ShHTL7 that are required for downstream signaling. Our findings identify Triton and the related 2‐[4‐(2,4,4‐trimethylpentan‐2‐yl)phenoxy]acetic acid as promising lead compounds for the rational design of efficient Striga‐specific herbicides.     

A new Chondrostoma species from the Büyük Menderes River Basin,Turkey (Teleostei: Cyprinidae)

In a study of the fishes of the Büyük Menderes River Basin, Aegean region of Turkey, two populations of Chondrostoma were found which showed clearly distinctive characters: the population from the Upper B. Menderes (I??kl? Lake) was attributed to C. meandrense Elvira, 1987, while the population from the Çine Stream in the Lower B. Menderes River basin proved to be a hitherto undescribed species: Chondrostoma turnai sp. n. Altogether 24 metric and 7 meristic parameters were compared. The new species is distinguished from C. meandrense and all other cogeners by a combination of the number of lateral line scales, the number of scale rows between the lateral line and the dorsal-fin origin, the number of scale rows of the lateral line and pelvic-fin origin, and the number of gill rakers on the first gill arch.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:811C213D-BEDD-4C8C-AE57-BFFA7964781A     

Degradation and transformation of anthracene by white-rot fungus Armillaria sp. F022

Characterization of anthracene metabolites produced by Armillaria sp. F022 was performed in the enzymatic system. The fungal culture was conducted in 100-mL Erlenmeyer flask containing mineral salt broth medium (20 mL) and incubated at 120 rpm for 5–30 days. The culture broth was then centrifuged at 10,000 rpm for 45 min to obtain the extract. Additionally, the effect of glucose consumption, laccase activity, and biomass production in degradation of anthracene were also investigated. Approximately, 92 % of the initial concentration of anthracene was degraded within 30 days of incubation. Dynamic pattern of the biomass production was affected the laccase activity during the experiment. The biomass of the fungus increased with the increasing of laccase activity. The isolation and characterization of four metabolites indicated that the structure of anthracene was transformed by Armillaria sp. F022 in two routes. First, anthracene was oxidized to form anthraquinone, benzoic acid, and second, converted into other products, 2-hydroxy-3-naphthoic acid and coumarin. Gas chromatography–mass spectrometry analysis also revealed that the molecular structure of anthracene was transformed by the action of the enzyme, generating a series of intermediate compounds such as anthraquinone by ring-cleavage reactions. The ligninolytic enzymes expecially free extracellular laccase played an important role in the transformation of anthracene during degradation period.