In situ mantle cell lymphoma: clinical implications of an incidental finding with indolent clinical behavior

Background Cyclin D1-positive B cells are occasionally found in the mantle zones of reactive lymphoid follicles, a condition that has been called 'in situ mantle cell lymphoma'. The clinical significance of this lesion remains uncertain. Design and Methods The clinical and pathological characteristics, including SOX11 expression, of 23 cases initially diagnosed as in situ mantle cell lymphoma were studied. Results Seventeen of the 23 cases fulfilled the criteria for in situ mantle cell lymphoma. In most cases, the lesions were incidental findings in reactive lymph nodes. The t(11; 14) was detected in all eight cases examined. SOX11 was positive in seven of 16 cases (44%). Five cases were associated with other small B-cell lymphomas. In two cases, both SOX11-positive, the in situ mantle cell lymphoma lesions were discovered after the diagnosis of overt lymphoma; one 4 years earlier, and one 3 years later. Twelve of the remaining 15 patients had a follow-up of at least 1 year (median 2 years; range, 1-19.5), of whom 11 showed no evidence of progression, including seven who were not treated. Only one of 12 patients with an in situ mantle cell lymphoma lesion and no diagnosis of mantle cell lymphoma at the time developed an overt lymphoma, 4 years later; this case was also SOX11-positive. The six remaining cases were diagnosed as mantle cell lymphoma with a mantle zone pattern. Five were SOX11-positive and four of them were associated with lymphoma without a mantle zone pattern. Conclusions In situ mantle cell lymphoma lesions are usually an incidental finding with a very indolent behavior. These cases must be distinguished from mantle cell lymphoma with a mantle zone pattern and overt mantle cell lymphoma because they may not require therapeutic intervention

Caenorhabditis elegans is a useful model for anthelmintic discovery

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery

Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial

BackgroundAnterior cruciate ligament (ACL) rupture is a common debilitating injury that can cause instability of the knee. We aimed to investigate the best management strategy between reconstructive surgery and non-surgical treatment for patients with a non-acute ACL injury and persistent symptoms of instability.MethodsWe did a pragmatic, multicentre, superiority, randomised controlled trial in 29 secondary care National Health Service orthopaedic units in the UK. Patients with symptomatic knee problems (instability) consistent with an ACL injury were eligible. We excluded patients with meniscal pathology with characteristics that indicate immediate surgery. Patients were randomly assigned (1:1) by computer to either surgery (reconstruction) or rehabilitation (physiotherapy but with subsequent reconstruction permitted if instability persisted after treatment), stratified by site and baseline Knee Injury and Osteoarthritis Outcome Score—4 domain version (KOOS4). This management design represented normal practice. The primary outcome was KOOS4 at 18 months after randomisation. The principal analyses were intention-to-treat based, with KOOS4 results analysed using linear regression. This trial is registered with ISRCTN, ISRCTN10110685, and ClinicalTrials.gov, NCT02980367.FindingsBetween Feb 1, 2017, and April 12, 2020, we recruited 316 patients. 156 (49%) participants were randomly assigned to the surgical reconstruction group and 160 (51%) to the rehabilitation group. Mean KOOS4 at 18 months was 73·0 (SD 18·3) in the surgical group and 64·6 (21·6) in the rehabilitation group. The adjusted mean difference was 7·9 (95% CI 2·5–13·2; p=0·0053) in favour of surgical management. 65 (41%) of 160 patients allocated to rehabilitation underwent subsequent surgery according to protocol within 18 months. 43 (28%) of 156 patients allocated to surgery did not receive their allocated treatment. We found no differences between groups in the proportion of intervention-related complications.InterpretationSurgical reconstruction as a management strategy for patients with non-acute ACL injury with persistent symptoms of instability was clinically superior and more cost-effective in comparison with rehabilitation management

EVA-1 Functions as an UNC-40 Co-receptor to Enhance Attraction to the MADD-4 Guidance Cue in <i>Caenorhabditis elegans</i>

<div><p>We recently discovered a secreted and diffusible midline cue called MADD-4 (an ADAMTSL) that guides migrations along the dorsoventral axis of the nematode <i>Caenorhabditis elegans</i>. We showed that the transmembrane receptor, UNC-40 (DCC), whose canonical ligand is the UNC-6 (netrin) guidance cue, is required for extension towards MADD-4. Here, we demonstrate that MADD-4 interacts with an EVA-1/UNC-40 co-receptor complex to attract cell extensions. EVA-1 is a conserved transmembrane protein with predicted galactose-binding lectin domains. EVA-1 functions in the same pathway as MADD-4, physically interacts with both MADD-4 and UNC-40, and enhances UNC-40's sensitivity to the MADD-4 cue. This enhancement is especially important in the presence of UNC-6. In EVA-1's absence, UNC-6 interferes with UNC-40's responsiveness to MADD-4; in UNC-6's absence, UNC-40's responsiveness to MADD-4 is less dependent on EVA-1. By enabling UNC-40 to respond to MADD-4 in the presence of UNC-6, EVA-1 may increase the precision by which UNC-40-directed processes can reach their MADD-4-expressing targets within a field of the UNC-6 guidance cue.</p></div

The novel nematicide wact-86 interacts with aldicarb to kill nematodes

<div><p>Parasitic nematodes negatively impact human and animal health worldwide. The market withdrawal of nematicidal agents due to unfavourable toxicities has limited the available treatment options. In principle, co-administering nematicides at lower doses along with molecules that potentiate their activity could mitigate adverse toxicities without compromising efficacy. Here, we screened for new small molecules that interact with aldicarb, which is a highly effective treatment for plant-parasitic nematodes whose toxicity hampers its utility. From our collection of 638 worm-bioactive compounds, we identified 20 molecules that interact positively with aldicarb to either kill or arrest the growth of the model nematode <i>Caenorhabditis elegans</i>. We investigated the mechanism of interaction between aldicarb and one of these novel nematicides called wact-86. We found that the carboxylesterase enzyme GES-1 hydrolyzes wact-86, and that the interaction is manifested by aldicarb’s inhibition of wact-86’s metabolism by GES-1. This work demonstrates the utility of <i>C</i>. <i>elegans</i> as a platform to search for new molecules that can positively interact with industrial nematicides, and provides proof-of-concept for prospective discovery efforts.</p></div

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in <i>Caenorhabditis elegans</i>

<div><p>The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in <i>Caenorhabditis elegans</i>, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in <i>madd-3</i> mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in <i>madd-3</i> mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.</p></div

MADD-4 attracts extending AVM axons via EVA-1 and UNC-40.

<p><b>A</b>. A schematic illustrating the area of the worm shown in B-D. Anterior is to the right and dorsal is up. <b>B–D</b>. Three worms expressing MADD-4::YFP from the dorsal muscles (from the <i>trIs78</i> transgenic array) in which the AVM axon (green arrowhead) extends ventrally (B), laterally (C) or dorsally (D). The ALMR neuron (yellow arrowhead) is also seen in all three panels. The <i>muIs32</i> transgene is used to visualize the AVM and ALMR neurons <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#pgen.1004521-Chng1" target="_blank">[22]</a>. The scale bar represents 50 µM. <b>E</b>. A genetic analysis of the AVM axon guidance errors in the indicated genetic background without transgenic expression of MADD-4. In all of these loss-of-function mutant backgrounds, the misguided axons invariably extend laterally. <b>F & G</b>. An analysis of laterally (F) or dorsally (G) -directed AVM axon extension in response to dorsally-expressed MADD-4::YFP. Shown is the percentage of animals in which the AVM extends laterally or dorsally, respectively. Note that in the background of dorsally-expressed MADD- 4, the AVM axon already extends dorsally in more than half of the <i>eva-1; unc-6</i> double mutant animals and leaves no room for the expected enhancement of <i>eva-1'</i>s lateral axon guidance defects by <i>unc-6</i>. Statistical significance is documented as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#pgen-1004521-g001" target="_blank">figure 1f</a>. The alleles used in this analysis are <i>madd-4(ok2854)</i>; <i>unc-40(n324), eva-1(ok1133), slt-1(ok255), and sax-3(ky200)</i>. Standard error of the mean is shown in all graphs.</p

EVA-1 interacts with UNC-40 via its transmembrane domain.

<p><b>A, A′</b> and <b>A″</b>. Shown is a single muscle cell that expresses EVA-1::CFP (expressed from an extra-chromosomal array) (<b>A</b>) that is enriched at muscle arm termini (arrowhead), and functional UNC-40::YFP (expressed from the <i>trIs34</i> array) (<b>A′</b>), and their co-localization (<b>A″</b>). The white arrow in the left-hand panels points to an example of muscle arm termini. The area that is boxed in each left-hand panel is respectively magnified in each right-hand panel. The arrow in the right-hand panels indicates one area of co-localization. The lack of dependence of EVA-1 and UNC-40 localization on the presence of UNC-40 and EVA-1, respectively, is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#pgen.1004521.s005" target="_blank">Figure S3a and S3b</a>. The scale bar represents 50 µM. <b>B</b>. A western blot showing that UNC-40 co-immunoprecipitates with full-length FLAG-tagged EVA-1, but not with a version of EVA-1 whose transmembrane domain has been swapped for that of PAT-2. Control blots showing a lack of interaction between EVA-1 and PAT-2 and between UNC-40 and PAT-2 are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#pgen.1004521.s005" target="_blank">Figure S3c and S3d</a>.</p

EVA-1 functions in a MADD-4 pathway to counteract UNC-6 interference.

<p><b>A</b>. An illustration of the worm that schematizes the muscle arms of ventral left distal body wall muscles. Anterior is to the left and dorsal is up. <b>B & C</b>. Micrographs showing a ventral view of a young adult worm of the indicated genotype with anterior to the left. The yellow arrowhead indicates the ventral cord, the white and green arrows indicates ventral left muscle numbers 11 and 19 (VL11 and VL19), respectively. The red arrowheads indicate the muscle arms of VL11 and VL19. The scale bar shows 50 µM. <b>D</b>. Quantification of muscle arm extension from VL11 and dorsal right muscle number 15 (DR15) in young adults of the indicated genotype. Dorsal muscle arm data is not shown for strains carrying the <i>unc-6</i> mutation because they lack motor neurons within the dorsal cord, which consequently confounds any interpretation of the resulting muscle arm phenotypes. In the last column, MADD-4 is over-expressed (O/E) pan-neuronally from the <i>trIs66</i> integrated transgenic array (see the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#s4" target="_blank">materials and methods</a> section for details). <b>E</b>. The average distance of the mid-proximal face of the indicated muscle to the ventral nerve cord in wild type animals (that also harbor the <i>trIs30</i> muscle arm and neuronal markers). <b>F</b>. Quantification of VL19 muscle arm extension in young adults of the indicated genotype. Statistical significance (<i>p</i><0.001) is indicated with a solid asterisk which is matched with a dot above the data point to which the comparison was made. Standard error of the mean is shown in all graphs.</p

EVA-1 functions cell-autonomously in muscles and interacts with MADD-4.

<p><b>A</b>. Muscle-expressed EVA-1::CFP rescues the muscle extension defects of <i>eva-1</i> mutants. <b>B</b>. A summary of EVA-1 domain function that is fully detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#pgen.1004521.s003" target="_blank">Figure S1</a>. <b>C&D</b>. FLAG-tagged receptors were expressed from HEK293 cells, bathed in conditioned media from other HEK293 cells that express HA- and Gaussia luciferase-tagged MADD-4 or SLT-1 ligands, and immunoprecipitated to determine the relative amounts of ligand that co-immunoprecipitates with the receptor (see the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#s4" target="_blank">materials and methods</a> section for more details). <b>C</b>. The western blot on the left shows the five immunoprecipitated FLAG-tagged receptors. The western blot on the right shows the two HA- and Gaussia luciferase-tagged ligands that were collected from cell culture. <b>D</b>. The normalized relative levels of luciferase signal that immunoprecipitated with each potential ligand-receptor complex. <b>E–I</b>. Shown are animals harbouring one of three different transgenes that drive the expression of either neuronally-expressed MADD-4::YFP (from the <i>trIs66</i> transgenic array) (<b>E</b>), muscle-expressed MADD-4::YFP (from the <i>trIs78</i> transgenic array) (<b>F</b>), or muscle-expressed EVA-1::CFP (from the <i>trIs89</i> transgenic array) (<b>G</b>), or animals harbouring two of the transgenes; <i>trIs66</i> and <i>trIs89</i> (<b>H</b>) and <i>trIs78</i> and <i>trIs89</i> (<b>I</b>). The relative levels of MADD-4::YFP expression from <i>trIs66</i> and <i>trIs78</i> is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#pgen.1004521.s004" target="_blank">Figure S2a</a>. Images show either the CFP channel (top), YFP channel (middle) or a merged view (bottom). Arrows in ‘H’ indicate the localization of MADD-4::YFP to EVA-1::CFP expressing muscles; arrows in ‘I’ indicate the vesicularization of MADD-4::YFP and EVA-1::CFP in the muscle cells. <b>J</b>. The quantification of neuronally-secreted MADD-4::YFP localization to muscles over-expressing the indicated receptor. <b>K</b>. The quantification of CFP vesicles in animals that over-express the indicated CFP-tagged receptors (x-axis) in muscles in either the presence of MADD-4::YFP expressed from dorsal muscles (mMADD-4) or pan-neuronally (nMADD-4). The colocalization of MADD-4 and EVA-1 with the RAB-11 and RAB-5 endosomal markers are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004521#pgen.1004521.s004" target="_blank">Figure S2b and S2c</a>. <b>L&M</b>. MADD-4::YFP fails to induce obvious vesicularization of UNC-40::CFP in a wild type background (L), but YFP-CFP vesicles are obvious in animals that lack UNC-6 (M). In A, J, and K, statistical significance (<i>p</i><0.05) is indicated with a solid asterisk which is matched with a dot above the data point to which the comparison was made. In all micrographs, the scale bar represents 50 micrometers. In all graphs, standard error of the mean is shown.</p