Three novel Ambrosia Fusarium Clade species producing multiseptate "dolphin-shaped" conidia, and an augmented description of Fusarium kuroshium.

The Ambrosia Fusarium Clade (AFC) is a monophyletic lineage within clade 3 of the Fusarium solani species complex (FSSC) that currently comprises 19 genealogically exclusive species. These fungi are known or predicted to be farmed by adult female Euwallacea ambrosia beetles as a nutritional mutualism (Coleoptera: Scolytinae; Xyleborini). To date, only eight of the 19 AFC species have been described formally with Latin binomials. We describe three AFC species, previously known as AF-8, AF-10, and AF-11, based on molecular phylogenetic analysis of multilocus DNA sequence data and comparative morphological/phenotypic studies. Fusarium duplospermum (AF-8) farmed by E. perbrevis on avocado in Florida, USA, is distinguished by forming two morphologically different types of multiseptate conidia and brownish orange colonies on potato dextrose agar (PDA). Fusarium drepaniforme (AF-10), isolated from an unknown woody host in Singapore and deposited as Herb IMI 351954 in the Royal Botanic Gardens, Kew, UK, under the name F. bugnicourtii, is diagnosed by frequent production of multiseptate sickle-shaped conidia. Fusarium papillatum (AF-11), isolated from mycangia of E. perbrevis infesting tea in Kandy, Sri Lanka, forms multiseptate clavate conidia that possess a papillate apical cell protruding toward the ventral side. Lastly, we prepared an augmented description of F. kuroshium (AF-12), previously isolated from the heads or galleries of E. kuroshio in a California sycamore tree, El Cajon, California, USA, and recently validated nomenclaturally as Fusarium. Conidia formed by F. kuroshium vary widely in size and shape, suggesting a close morphological relationship with F. floridanum, compared with all other AFC species. Maximum likelihood and maximum parsimony analyses of a multilocus data set resolve these three novel AFC species, and F. kuroshium, as phylogenetically distinct based on genealogical concordance. Given the promiscuous nature of several Euwallacea species, and the overlapping geographic range of several AFC species and Euwallacea ambrosia beetles, the potential for symbiont switching among sympatric species is discussed.

Rat embryonic fibroblasts immortalized by MRPS18-2 protein are target for NK-cells.

Overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts (REFs). The derived cells (18IM) expressed embryonic stem cell markers. Noteworthy, genes encoding the COX family proteins were up-regulated significantly. It is known that the COX family proteins are involved in the regulation of immune response. In the present work we demonstrate that 18IM cells behave like stem cells when subjected to directed differentiation in vitro. However, unlike stem cells, 18IM cells do not develop tumors in vivo, in SCID mice. This phenomenon is observed due to the strong natural killer (NK) cell immunogenicity. 18IM cells were better recognized by NK cells, compared with primary REFs, as was shown by a standard NK killing assay. Our data explain asymmetry in behavior of stem-like cells in vivo and in vitro, and this support the notion that stem and/or cancer-initiating cells are preferred targets for NK-cells. Concluding, the S18-2 protein is a putative target for cancer vaccines.

HLA class I downregulation is associated with enhanced NK-cell killing of melanoma cells with acquired drug resistance to BRAF inhibitors.

The frequent development of drug resistance to targeted therapies in cancer patients has stimulated interest in strategies counteracting resistance. Combining immunotherapies with targeted therapies is one such strategy. In this context, we asked whether human NK cells can target melanoma cells that have acquired resistance to selective inhibitors targeting activating mutants of the B-Raf kinase (BRAF inhibitors, BRAFi). We generated drug-resistant cell variants in vitro from human BRAF-mutant melanoma cell lines MEL-HO, COLO-38, SK-MEL-37, 1520 and from primary melanoma cells freshly isolated from two patients. All drug-resistant cell variants remained susceptible to lysis by IL-2-activated NK cells; and two BRAFi-resistant lines (BRAFi-R) became significantly more susceptible to NK-cell lysis than their parental lines. This was associated with significant HLA class I antigen downregulation and PD-L1 upregulation on the drug-resistant lines. Although blocking HLA class I enhanced the extent of lysis of both BRAFi-R and parental cells to NK-cell-mediated lysis, antibody-mediated inhibition of PD1-PD-L1 interactions had no detectable effect. HLA class I antigen expression on BRAFi-R melanoma variants thus appears to play a major role in their susceptibility to NK-cell cytotoxicity. These findings suggest that NK-cell-based immunotherapy may be a viable approach to treat melanoma patients with acquired resistance to BRAF inhibitors.

Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism.

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1-12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea-Fusarium mutualism near the Oligocene-Miocene boundary ∼19-24 Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.

Human NK cells selective targeting of colon cancer-initiating cells: a role for natural cytotoxicity receptors and MHC class I molecules.

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

A preliminary study of locognosia in people affected by leprosy.

Locognosia is the ability to localize a sensory stimulus on the body's surface and can be tested by graded filaments (Semmes-Weinstein monofilaments). This point localization of sensation (locognosia) wastested by SW filaments over four quadrants of the pulp of the fingers in ulnar/median and ulnar paralysis in 38 new patients affected by leprosy. The results were compared with standard testing of sensation at selected sites by Semmes Weinstein monofilament. Both pulp quadrant testing and standard site testing were done in leprosy patients and also in a group of controls. Sensation was tested in 73 hands in leprosy patients and 34 hands in controls. Results indicate a positive correlation between locognosia and standard SW filament testing. When locognosia and standard SW filament tests were compared, there was significant difference between the two tests to pick up abnormal sensation in leprosy patients both over the entire hand and over individual fingers. This preliminary study suggests that locognosia may be a useful tool to diagnose sensory impairment in leprosy. Further studies are required to corroborate this.

Exobasidium vexans infection of Camellia sinensis increased 2,3-cis isomerisation and gallate esterification of proanthocyanidins.

Infection of leaves of tea (Camellia sinensis (Kuntze) L, cv TRI 2025) which was susceptible to blister blight (Exobasidium vexans Massee), resulted in a shift of the proanthocyanidin stereochemistry away from 2,3-trans (e.g. catechin and gallocatechin) and towards 2,3-cis (e.g. epicatechin and epigallocatechin). Infection also resulted in increased gallic acid esterification of the initiating subunits of proanthocyanidins. This was shown by both mass spectroscopy and phloroglucinolysis. Proanthocyanidins isolated from healthy tissue had a predominantly 2,3-trans stereochemistry which accounted for 53% and 61% of the total initiating and extension units of proanthocyanidin, respectively. Conversely in infected tissue, proanthocyanidin subunits with a 2,3-trans stereochemistry accounted for 26% and 40% of the total initiating and extension units, respectively. Infection had little impact on the hydroxylation state of the B-rings of proanthocyanidins. The products of acid hydrolysis under oxidative conditions had a slight excess of di-hydroxylated B-rings with cyanidin accounting for 58.3+/-0.05% and 60.4+/-0.2% of the total anthocyanidin recovered following hydrolysis of proanthocyanidin isolated from infected and healthy leaves, respectively. Similar results were obtained by phloroglucinolysis.