Autologous stem cell transplantation for untreated transformed indolent B-cell lymphoma in first remission: an international, multi-centre propensity-score-matched study.

High-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT) are used as consolidation in first remission (CR1) in some centres for untreated, transformed indolent B-cell lymphoma (Tr-iNHL) but the evidence base is weak. A total of 319 patients with untreated Tr-iNHL meeting prespecified transplant eligibility criteria [age <75, LVEF ≥45%, no severe lung disease, CR by positron emission tomography or computed tomography ≥3 months after at least standard cyclophosphamide, doxorubicin, vincristine and prednisolone with rituximab (R-CHOP) intensity front-line chemotherapy] were retrospectively identified. Non-diffuse large B-cell lymphoma transformations were excluded. About 283 (89%) patients had follicular lymphoma, 30 (9%) marginal-zone lymphoma and six (2%) other subtypes. Forty-nine patients underwent HDC/ASCT in CR1, and a 1:2 propensity-score-matched cohort of 98 patients based on age, stage and high-grade B-cell lymphoma with MYC, BCL2 and/or BCL6 rearrangements (HGBL-DH) was generated. After a median follow-up of 3·7 (range 0·1-18·3) years, ASCT was associated with significantly superior progression-free survival [hazard ratio (HR) 0·51, 0·27-0·98; P = 0·043] with a trend towards inferior overall survival (OS; HR 2·36;0·87-6·42; P = 0·1) due to more deaths from progressive disease (8% vs. 4%). Forty (41%) patients experienced relapse in the non-ASCT cohort - 15 underwent HDC/ASCT with seven (47%) ongoing complete remission (CR); 10 chimeric antigen receptor-modified T-cell (CAR-T) therapy with 6 (60%) ongoing CR; 3 allogeneic SCT with 2 (67%) ongoing CR. Although ASCT in CR1 improves initial duration of disease control in untreated Tr-iNHL, the impact on OS is less clear with effective salvage therapies in this era of CAR-T.

Rapid detection of Phytophthora ramorum and P. kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device.

ABSTRACT A method for nucleic-acid-based detection of pathogens in plant material has been developed which comprises a simple and rapid method for extracting DNA on the nitrocellulose membranes of lateral-flow devices, loop-mediated isothermal amplification (LAMP) of target DNA using labeled primers, and detection of the generically labeled amplification products by a sandwich immunoassay in a lateral-flow-device format. Each of these steps can be performed without specialist equipment and is suitable for on-site use, and a result can be obtained in just over an hour. A LAMP assay for the detection of plant DNA (cytochrome oxidase gene) can be used in conjunction with pathogen-specific assays to confirm negative results. The use of this method is demonstrated for the detection of Phytophthora ramorum, the causal agent of sudden oak death and dieback/leaf blight in a range of tree, shrub, and herbaceous species, and the recently described pathogen P. kernoviae.

Detection of Botrytis cinerea by loop-mediated isothermal amplification.

AIMS: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop-mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. METHODS AND RESULTS: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross-reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real-time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. CONCLUSIONS: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: The LAMP method combines the sensitivity and specificity of nucleic acid-based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on-site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.

Anxiety and depression may have an evolutionary role as negative reinforcers, encouraging socialisation.

Anxiety and depression are both very common mental states and both are quite unpleasant. Their high prevalence and preservation make it likely they have considerable evolutionary significance and in some way improve the chances of an individual's survival. The following article proposes that much of the anxiety and depression we experience is primarily acting as a negative reinforcer, encouraging socialisation. These feelings are experienced most intensely when we are isolated and have evolved to discourage this highly maladaptive behaviour, there being major advantages for being with other people. Particular problems are considered, including aspects of autism and alcohol and tranquilliser abuse. How the presence of other people may alter the expression of anxiety and depression is considered, in addition to implications for psychiatry.

Close linkage between the Cf-2/Cf-5 and Mi resistance loci in tomato.

Analysis of restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers in tomato plants segregating for resistance to the fungus Cladosporium fulvum was used to localize the resistance genes Cf-2 and Cf-5 to the same region of chromosome 6. This region, between GP79 and Aps-1, is the same as that reported for the Mi gene, which confers resistance to root-knot nematodes (meloidogyne spp.). Recombination values based on F2 populations from crosses between near-isogenic lines of L. esculentum 'Moneymaker' carrying Cf-2 or Cf-5 and Lycopersicon pennellii, indicate that this region occupies 4-5 centiMorgans (cM). However, in F2 populations from crosses between the L. esculentum stock LA1190 carrying yv and these lines, this value is 1-2 cM. The Cf-2 gene, introduced into L. esculentum from L. pimpinellifolium, is on an introgressed segment that extends from a point distal to GP79 to a point between TG232 and H2D1. The origin of Cf-5 was found to be L. esculentum var. cerasiforme rather than L. pimpinellifolium as previously reported. No RFLP markers and only one RAPD marker showed a polymorphism between Moneymaker and the near-isogenic line carrying Cf-5.

Cloning of the CYP51 gene from the eyespot pathogen Tapesia yallundae indicates that resistance to the DMI fungicide prochloraz is not related to sequence changes in the gene encoding the target site enzyme.

Resistance to sterol 14 alpha-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14 alpha-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.

Loop-mediated isothermal amplification for rapid detection of the causal agents of cassava brown streak disease.

The causal agents of cassava brown streak disease have recently been identified as Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Primers have been developed for rapid detection of these viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP). Performance of the RT-LAMP assays compared favourably with published RT-PCR and real-time RT-PCR methods. Furthermore, amplification by RT-LAMP is completed in 40 min and does not require thermal cycling equipment. Modification of the RT-LAMP reactions to use labelled primers allowed rapid detection of amplification products using lateral flow devices containing antibodies specific to the incorporated labels, avoiding the need for fluorescence detection or gel electrophoresis.

Integration of a temperate phage infecting Spiroplasma citri.

A physical map of the genome of a temperate Type 3 spiroplasma-virus, ai, has been constructed. Host DNA has been digested with restriction enzymes, and recombinant DNA clones of ai fragments in coliphage M13 vectors have been used as probes to detect viral DNA sequences integrated into spiroplasmas. All strains of Spiroplasma citri examined contained a deleted form of ai integrated as a cryptic prophage which was unable to confer resistance to ai superinfection. Stable ai lysogens also contained a complete ai genome. We propose that the infecting viral DNA circularizes and integrates adjacent to the cryptic prophage in a site-specific recombination event at unique points in both the virus and host genomes.

Lysogenisation of Spiroplasma citri by a type 3 spiroplasmavirus.

A short-tailed polyhedral spiroplasmavirus, ai, with a plaque morphology typical of temperate phages and a linear double-stranded DNA genome which can circularise due to the presence of cohesive ends, was able to lysogenise Spiroplasma citri. Lysogenic sporoplasmas spontaneously released ai virus at a low level, and were immune to superinfection by the released virus, but not to infection by two serologically related viruses. These properties were retained following repeated cultivation in virus antiserum. Viral sequences were detectable in host DNA by probing with recombinant DNA clones of ai fragments in coliphage M13 vectors. All strains of S. citri examined contained a deleted form of ai integrated as a cryptic prophage. We propose that ai circularises and integrates into the host genome adjacent to the cryptic prophage by a site-specific recombination event. Evidence of a correlation between lysogenisation and the attenuation of pathogenicity is presented.

Nucleotide sequence relationships of double-stranded RNAs in flax rust, Melampsora lini.

Flax rust, Melampsora lini strain SP6, contains 11 double-stranded (ds) RNA molecules with a total length of about 25 kbp. The dsRNAs are inherited in three genetic units: the L unit comprising a single 5.2 kbp dsRNA and contained within a 40-nm virus-like particle, and the A and B units each consisting of five dsRNAs (A1-A5, and B1-B5) ranging in size from 1.2 to 2.7 kbp. This paper reports the isolation of a cDNA library representing 10 of the 11 dsRNAs. By nucleic-acid hybridization techniques it has been shown that all ten sequences are unique showing no detectable cross-hybridization with any other dsRNA present in the rust. A near full-length sequence of 1932 bp of the B3 dsRNA is reported and contains several open reading frames, the largest of which comprises most of the molecule.

Characterisation of a virus infecting the wall-free prokaryote Spiroplasma citri.

A short-tailed polyhedral virus (ai) with a plaque morphology typical of temperate phages has been purified from the wall-less prokaryote Spiroplasma citri. Adsorption of the virus to host cells and isolated membranes was dependent on pH and divalent cation concentration. One-step growth curves and premature lysis experiments coupled with immunodetection of intracellular viral antigens showed a steady accumulation of viral products from 2 hr after infection. Mature virus was present after 3-4 hr. Virus release started after 5-6 hr and continued for a further 4-5 hr. The virus had antigens in common with other type 3 spiroplasma viruses and purified DNA hybridised to specific pieces of DNA from these related viruses. A physical map of the virus genome has been constructed. Unlike those of related viruses, the genome of ai can form noncovalently closed circles and multimers. The significance of this in relation to the biological properties of the virus is discussed.

Expression of the beta-thalassemia gene in the first trimester fetus.

To determine whether beta-thalassemia can be detected in the fetus, blood was obtained from abortuses of normal mothers and of mothers with beta-thalassemia trait. The red cells were incubated with radioactive leucine and the globin chains were analyzed by radiochromatography. Two independent methods were utilized to correct the results for contamination by maternal radioactive beta-chain, and the corrected beta/gamma ratios were compared to a previously established range of normal fetal beta/gamma synthetic ratios obtained by similar measurements in pure fetal cells. In the erythroid cells of three fetuses from mothers with beta-thalassemia trait, the beta/gamma synthetic ratio was normal in two. The third had a beta/gamma ratio of 0.04 at 10 1/2 weeks, a 50% reduction, consistent with fetal beta-thalassemia trait. Two other fetuses, derived from parents both of whom had beta-thalassemia trait, were also studied. One had a beta/gamma ratio of 0.029 at 8 weeks, a 65% reduction, also consistent with beta-thalassemia trait. The cells of the other had a ratio of essentially zero at 11 weeks, highly suggestive of homozygous beta-thalassemia. Although further experience will be needed to distinguish the homozygous and heterozygous states reliably, it now appears that the beta-thalassemia gene is expressed in the first trimester. Therefore these data suggest that the antenatal diagnosis of beta-thalassemia is becoming an attainable goal.