The multi-specific VH-based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity.

BACKGROUND: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. METHODS: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. RESULTS: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. CONCLUSIONS: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

Global Cropland Connectivity: A Risk Factor for Invasion and Saturation by Emerging Pathogens and Pests.

The geographic pattern of cropland is an important risk factor for invasion and saturation by crop-specific pathogens and arthropods. Understanding cropland networks supports smart pest sampling and mitigation strategies. We evaluate global networks of cropland connectivity for key vegetatively propagated crops (banana and plantain, cassava, potato, sweet potato, and yam) important for food security in the tropics. For each crop, potential movement between geographic location pairs was evaluated using a gravity model, with associated uncertainty quantification. The highly linked hub and bridge locations in cropland connectivity risk maps are likely priorities for surveillance and management, and for tracing intraregion movement of pathogens and pests. Important locations are identified beyond those locations that simply have high crop density. Cropland connectivity risk maps provide a new risk component for integration with other factors-such as climatic suitability, genetic resistance, and global trade routes-to inform pest risk assessment and mitigation.

A method for generating virus-free cassava plants to combat viral disease epidemics in Africa.

Here, we report a method to clean cassava plants from viral infections that cause cassava mosaic and brown streak diseases in Africa. Infected plants of resistant or tolerant varieties from Malawi, Mozambique, Kenya, Tanzania and Uganda were cleaned in the UK using a combination of tissue culture, chemotherapy and thermotherapy. In the first cycle of our virus-indexing procedure, we successfully cleaned 27 of the 31 varieties (87%), and after an additional three cleaning cycles, all plants were virus-free. Virus-free tissue-cultured plants were shipped back to Africa for distribution to farmers. This first cross-boundary effort provides important lessons for mitigating the two-major cassava viral diseases.

Assessing the Degeneration of Cassava Under High-Virus Inoculum Conditions in Coastal Tanzania.

Cassava brown streak disease (CBSD), caused by cassava brown streak ipomoviruses (CBSIs), has become the most debilitating biotic stress to cassava production in East and Central Africa. Lack of CBSD-resistant varieties has necessitated the search for alternative control measures. Most smallholder farmers reuse stems from previous crops for planting in the new season. Recycling planting material in this way can lead to "degeneration" owing to the compounding effects of disease. In this study, degeneration was defined as the increase in CBSD incidence and reduction in marketable root yield over time. An experiment was established to study the rates of degeneration in selected cassava varieties Chereko, KBH2002_135, Kipusa, Kizimbani, and Mkuranga1 and cultivars Kiroba and Kikombe under high-CBSD inoculum conditions in Bagamoyo, Tanzania from 2013 to 2017. The experiment was replicated across two seasons: the first planted during the long rains (Masika) between March and June and the second planted during the short rains (Vuli) between October and December. Mean abundance of the whitefly vector (Bemisia tabaci) was much greater during the Vuli season (>19 insects per plant) than the Masika season (<2 insects per plant). CBSD shoot symptoms occurred naturally and were observed only on Kikombe, Kiroba, and Kipusa. New materials had overall lower CBSD shoot incidences (1.5%) compared with recycled materials (6.9%) in Masika, although no significant differences were obvious in Vuli. However, Masika (8.7%) had an overall lower CBSD shoot incidence than Vuli (16.5%) in the varieties that had shoot symptoms. CBSD root incidences were higher in Vuli (10.3%) than Masika (4.4%), and root yields in Masika (29.4 t/ha) were significantly greater than those in Vuli (22.5 t/ha). The highest percentage of roots rendered unusable owing to CBSD was observed in Vuli. There was significantly higher unusable root incidence in recycled materials (3.7%) than in new materials (1.4%) in Masika but not in Vuli. Overall root yield was similar between recycled and new materials in either season. Significant reductions in root yield over the course of the experiment were observed both in Masika and Vuli, whereas changes in marketable yield were significant only in Masika. Differences in the response of varieties to degeneration led to the identification of four degeneration patterns, namely "strong," "moderate," "mild," and "delayed" degeneration. The strongest effects of degeneration were most obvious in the susceptible cultivar (Kikombe), which also had the lowest marketable yield in either season. Seasonal differences were a key driver of degeneration, because its effects were much greater in Vuli than Masika. To the best of our knowledge, this work reports the first study of degeneration caused by cassava viruses.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Two Novel DNAs That Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease.

UNLABELLED: Cassava mosaic begomoviruses (CMBs) cause cassava mosaic disease (CMD) across Africa and the Indian subcontinent. Like all members of the geminivirus family, CMBs have small, circular single-stranded DNA genomes. We report here the discovery of two novel DNA sequences, designated SEGS-1 and SEGS-2 (forsequencesenhancinggeminivirussymptoms), that enhance symptoms and break resistance to CMD. The SEGS are characterized by GC-rich regions and the absence of long open reading frames. Both SEGS enhanced CMD symptoms in cassava (Manihot esculentaCrantz) when coinoculated withAfrican cassava mosaic virus(ACMV),East African cassava mosaic Cameroon virus(EACMCV), orEast African cassava mosaic virus-Uganda(EACMV-UG). SEGS-1 also overcame resistance of a cassava landrace carrying the CMD2 resistance locus when coinoculated with EACMV-UG. Episomal forms of both SEGS were detected in CMB-infected cassava but not in healthy cassava. SEGS-2 episomes were also found in virions and whiteflies. SEGS-1 has no homology to geminiviruses or their associated satellites, but the cassava genome contains a sequence that is 99% identical to full-length SEGS-1. The cassava genome also includes three sequences with 84 to 89% identity to SEGS-2 that together encompass all of SEGS-2 except for a 52-bp region, which includes the episomal junction and a 26-bp sequence related to alphasatellite replication origins. These results suggest that SEGS-1 is derived from the cassava genome and facilitates CMB infection as an integrated copy and/or an episome, while SEGS-2 was originally from the cassava genome but now is encapsidated into virions and transmitted as an episome by whiteflies. IMPORTANCE: Cassava is a major crop in the developing world, with its production in Africa being second only to maize. CMD is one of the most important diseases of cassava and a serious constraint to production across Africa. CMD2 is a major CMD resistance locus that has been deployed in many cassava cultivars through large-scale breeding programs. In recent years, severe, atypical CMD symptoms have been observed occasionally on resistant cultivars, some of which carry the CMD2 locus, in African fields. In this report, we identified and characterized two DNA sequences, SEGS-1 and SEGS-2, which produce similar symptoms when coinoculated with cassava mosaic begomoviruses onto a susceptible cultivar or a CMD2-resistant landrace. The ability of SEGS-1 to overcome CMD2 resistance and the transmission of SEGS-2 by whiteflies has major implications for the long-term durability of CMD2 resistance and underscore the need for alternative sources of resistance in cassava.

Survey, Molecular Detection, and Characterization of Geminiviruses Associated with Cassava Mosaic Disease in Zambia.

A survey was conducted from April to May 2014 in 214 farmers' fields located across six major cassava-producing provinces (Western, Northwestern, Northern, Luapula, Lusaka, and Eastern) of Zambia to determine the status of cassava mosaic disease (CMD) and the species diversity of associated cassava mosaic geminiviruses (CMG). Mean CMD incidence varied across all six provinces but was greatest in Lusaka Province (81%) and least in Northern Province (44%). Mean CMD severity varied slightly between provinces, ranging from 2.78 in Eastern Province to 3.00 in Northwestern Province. Polymerase chain reaction discrimination of 226 survey samples, coupled with complete DNA-A genome sequence analysis, revealed the presence of African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), and East African cassava mosaic Malawi virus (EACMMV) as single or mixed infections of different proportions. Single-virus infections were predominant, occurring in 62.8% (ACMV), 5.8% (EACMMV), and 2.2% (EACMV) of samples relative to mixed-virus infections, which occurred in 19.5% (ACMV + EACMMV), 0.4% (ACMV + EACMV), and 0.9% (ACMV + EACMV + EACMMV) of samples. Phylogenetic analysis revealed the segregation of virus isolates from Zambia into clades specific to ACMV, EACMV, and EACMMV, further confirming the presence of all three viruses in Zambia. The results point to a greater diversity of CMG across major cassava-growing provinces of Zambia and implicate contaminated cassava cuttings in disease spread.

Cassava brown streak disease: a threat to food security in Africa.

Cassava brown streak disease (CBSD) has emerged as the most important viral disease of cassava (Manihot esculenta) in Africa and is a major threat to food security. CBSD is caused by two distinct species of ipomoviruses, Cassava brown streak virus and Ugandan cassava brown streak virus, belonging to the family Potyviridae. Previously, CBSD was reported only from the coastal lowlands of East Africa, but recently it has begun to spread as an epidemic throughout the Great Lakes region of East and Central Africa. This new spread represents a major threat to the cassava-growing regions of West Africa. CBSD-resistant cassava cultivars are being developed through breeding, and transgenic RNA interference-derived field resistance to CBSD has also been demonstrated. This review aims to provide a summary of the most important studies on the aetiology, epidemiology and control of CBSD and to highlight key research areas that need prioritization.

Th1 cytokines are more effective than Th2 cytokines at licensing anti-tumour functions in CD40-activated human macrophages in vitro.

CD40 agonists are showing activity in early clinical trials in patients with advanced cancer. In animal models, CD40 agonists synergise with T-cell-activating therapies to inhibit tumour growth by driving tumour macrophage repolarisation from an immunosuppressive to a Th1 immunostimulatory, tumouricidal phenotype. We therefore tested the hypothesis that T-cell-derived cytokines license anti-tumour functions in CD40-activated human macrophages. CD40 ligand (CD40L) alone activated macrophages to produce immunosuppressive IL-10, in a similar fashion to bacterial LPS, but failed to promote anti-tumour functions. The Th1 cytokine IFN-γ optimally licensed CD40L-induced macrophage anti-tumour functions, inducing a switch from IL-10 to IL-12p70 production, promoting macrophage-mediated Th1 T-cell skewing and enhancing tumouricidal activity. We found that even the Th2 cytokines IL-4 and IL-13 promoted IL-12p70 production (albeit without inhibiting IL-10 production) and enhanced Th1 T-cell skewing by CD40L-activated macrophages. However, IL-4 and IL-13 did not enhance tumouricidal activity in CD40L-activated macrophages. Thus, while both Th1 and Th2 cytokines biased macrophages to a Th1 immunostimulatory phenotype, only Th1 cytokines promoted tumouricidal activity in CD40L-activated macrophages. The presence of tumour-infiltrating Th1 or Th2 cells might therefore be predictive for patient response to CD40 agonism.

Design of Crosslinking Antibodies For T-Cell Activation: Experimental and Computational Analysis of PD-1/CD137 Bispecific Agents.

Bispecific and multispecific agents have become increasingly utilized in cancer treatment and immunotherapy, yet their complex design parameters present a challenge in developing successful therapeutics. Bispecifics that crosslink receptors on two opposing cells can provide specific activation of a receptor only when these cells are in close spatial proximity, such as an immune cell and cancer cell in a tumor. These agents, including T cell activating bispecifics, can avoid off-tumor toxicity through activation only in the tumor microenvironment by utilizing a tumor target to cluster T-cell receptors for a selective costimulatory signal. Here, we investigate a panel of PD-1/CD137 targeted Humabody VH domains to determine the key factors for T cell activation, such as affinity, valency, expression level, domain orientation, and epitope location. Target expression is a dominant factor determining both specificity and potency of T cell activation. Given an intrinsic expression level, the affinity can be tuned to modulate the level of activation and IC50 and achieve specificity between low and high expression levels. Changing the epitope location and linker length showed minor improvements to activation at low expression levels, but increasing the valency for the target decreased activation at all expression levels. By combining non-overlapping epitopes for the target, we achieved higher receptor activation at low expression levels. A kinetic model was able to capture these trends, offering support for the mechanistic interpretation. This work provides a framework to quantify factors for T cell activation by cell-crosslinking bispecific agents and guiding principles for the design of new agents.

Impact of tissue penetration and albumin binding on design of T cell targeted bispecific agents.

Bispecific agents are a rapidly growing class of cancer therapeutics, and immune targeted bispecific agents have the potential to expand functionality well beyond monoclonal antibody agents. Humabodies⁎ are fully human single domain antibodies that can be linked in a modular fashion to form multispecific therapeutics. However, the effect of heterogeneous delivery on the efficacy of crosslinking bispecific agents is currently unclear. In this work, we utilize a PSMA-CD137 Humabody with an albumin binding half-life extension (HLE) domain to determine the impact of tissue penetration on T cell activating bispecific agents. Using heterotypic spheroids, we demonstrate that increased tissue penetration results in higher T cell activation at sub-saturating concentrations. Next, we tested the effect of two different albumin binding moieties on tissue distribution using albumin-specific HLE domains with varying affinities for albumin and a non-specific lipophilic dye. The results show that a specific binding mechanism to albumin does not influence tissue penetration, but a non-specific mechanism reduced both spheroid uptake and distribution in the presence of albumin. These results highlight the potential importance of tissue penetration on bispecific agent efficacy and describe how the design parameters including albumin-binding domains can be selected to maximize the efficacy of bispecific agents.

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

PURPOSE: CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL DESIGN: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Cutting Dipping Application of Flupyradifurone against Cassava Whiteflies Bemisia tabaci and Impact on Its Parasitism in Cassava.

The cassava whitefly Bemisia tabaci causes damage in cassava through the feeding and vectoring of plant viruses that cause cassava mosaic and cassava brown streak diseases. This study sought to explore the efficacy of cutting dipping in flupyradifurone for whitefly control and the impact of the mode of application on whitefly parasitism under farmer field conditions. The insecticide treatment significantly reduced adult whiteflies by 41%, nymphs by 64%, and cassava mosaic disease (CMD) incidence by 16% and increased root yield by 49%. The whitefly parasitism rate by Encarsia spp. parasitoids was 27.3 and 21.1%, while Eretmocerus spp. had 26.7 and 18.0% in control and flupyradifurone, respectively, and these differences were not significant. Electropenetrography recordings of whitefly feeding behaviour on flupyradifurone-treated plants showed significantly reduced probing activity and a delay in reaching the phloem as compared to the control. The findings from this study demonstrated that cassava cutting dipping in flupyradifurone significantly reduces whitefly numbers and cassava mosaic disease incidence, thus contributing to a significant root yield increase in cassava. Flupyradifurone applied through cutting dips does not significantly impact parasitism rates in cassava fields. Routine monitoring of parasitoids and predators in insecticide-treated versus control fields should be emphasized to determine the impact of pesticides on these beneficial non-target organisms.

Influence of altitude as a proxy for temperature on key Musa pests and diseases in watershed areas of Burundi and Rwanda.

Pests and diseases are key biotic constraints limiting banana production among smallholder farmers in Eastern and Central Africa. Climate changemay favour pest and disease development and further exacerbate the vulnerability of smallholder farming systems to biotic constraints. Information on effects of climate change on pests and pathogens of banana is required byby policy makers and researchers in designing control strategies and adaptation plans. Since altitude is inversely related to temperature, this study used the occurrence of key banana pests and diseases along an altitude gradient as a proxy for the potential impact of changes in temperature associated with global warming on pests and diseases. We assessed the occurrence of banana pests and diseases in 93 banana fields across three altitude ranges in Burundi and 99 fields distributed in two altitude ranges in Rwanda watersheds. Incidence and prevalence of Banana Bunchy Top Disease (BBTD) and Fusarium wilt (FW) was significantly associated with temperature and altitude in Burundi, revealing that increasing temperatures may lead to upward movement of banana diseases. No significant associations with temperature and altitude were observed for weevils, nematodes and Xanthomonas wilt of banana (BXW). Data collected in this study provides a baseline to verify and guide modelling work to predict future pest and disease distribution according to climate change scenarios. Such information is useful in informing policy makers and designing appropriate management strategies.

Evaluation of the Efficacy of Flupyradifurone against Bemisia tabaci on Cassava in Tanzania.

A novel butenolide insecticide-flupyradifurone (Sivanto SL 200)-was evaluated for efficacy against cassava-colonizing Bemisia tabaci whitefly under laboratory, screenhouse and field conditions. LC50 values from leaf disc spray assays were comparable for both flupyradifurone (12.7 g a.i/100 L) and imidacloprid (12.6 g a.i/100 L). Both insecticides caused high levels of adult whitefly mortality in leaf disc and leaf dip assays when compared to untreated controls. In screenhouse-based trials, longer soaking (60 min) with flupyradifurone or imidacloprid was more effective than shorter soaking durations (15 or 30 min). In field spraying experiments, flupyradifurone significantly reduced whiteflies, and both insecticides demonstrated powerful knockdown effects on whitefly adult abundances over a period up to 24 h. Single cutting dip application of flupyradifurone reduced whitefly adult abundance by 2 to 6 times, and nymphs by 2 to 13 times. Lower whitefly abundances resulting from insecticide application reduced the incidence of CMD or CBSD. In addition, in field experiments, whiteflies were fewer during the long rainy season (Masika) and on cassava variety Mkuranga1. The findings from this study demonstrate that cutting dips with flupyradifurone could be incorporated as a management tactic against cassava whiteflies. This would ideally be combined in an IPM strategy with other cassava virus and virus vector management tactics including host-plant resistance, phytosanitation and the use of clean seed.

Early detection of plant virus infection using multispectral imaging and spatial-spectral machine learning.

Cassava brown streak disease (CBSD) is an emerging viral disease that can greatly reduce cassava productivity, while causing only mild aerial symptoms that develop late in infection. Early detection of CBSD enables better crop management and intervention. Current techniques require laboratory equipment and are labour intensive and often inaccurate. We have developed a handheld active multispectral imaging (A-MSI) device combined with machine learning for early detection of CBSD in real-time. The principal benefits of A-MSI over passive MSI and conventional camera systems are improved spectral signal-to-noise ratio and temporal repeatability. Information fusion techniques further combine spectral and spatial information to reliably identify features that distinguish healthy cassava from plants with CBSD as early as 28 days post inoculation on a susceptible and a tolerant cultivar. Application of the device has the potential to increase farmers' access to healthy planting materials and reduce losses due to CBSD in Africa. It can also be adapted for sensing other biotic and abiotic stresses in real-world situations where plants are exposed to multiple pest, pathogen and environmental stresses.

Epidemiological Analysis of Cassava Mosaic and Brown Streak Diseases, and Bemisia tabaci in the Comoros Islands.

A comprehensive assessment of cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) was carried out in Comoros where cassava yield (5.7 t/ha) is significantly below the African average (8.6 t/ha) largely due to virus diseases. Observations from 66 sites across the Comoros Islands of Mwali, Ngazidja, and Ndzwani revealed that 83.3% of cassava fields had foliar symptoms of CBSD compared with 95.5% for CMD. Molecular diagnostics confirmed the presence of both cassava brown streak ipomoviruses (CBSIs) and cassava mosaic begomoviruses (CMBs). Although real-time RT-PCR only detected the presence of one CBSI species (Cassava brown streak virus, CBSV) the second species (Ugandan cassava brown streak virus, UCBSV) was identified using next-generation high-throughput sequencing. Both PCR and HTS detected the presence of East African cassava mosaic virus (EACMV). African cassava mosaic virus was not detected in any of the samples. Four whitefly species were identified from a sample of 131 specimens: Bemisia tabaci, B. afer, Aleurodicus dispersus, and Paraleyrodes bondari. Cassava B. tabaci comprised two mitotypes: SSA1-SG2 (89%) and SSA1-SG3 (11%). KASP SNP genotyping categorized 82% of cassava B. tabaci as haplogroup SSA-ESA. This knowledge will provide an important base for developing and deploying effective management strategies for cassava viruses and their vectors.

Diversity and Distribution of Whiteflies Colonizing Cassava in Eastern Democratic Republic of Congo.

The present study characterizes Bemisia tabaci and Bemisia afer from cassava in eastern Democratic Republic of Congo (DRC). The Mitochondrial COI sequencing revealed the occurrence of six cassava B. tabaci mitotypes, which were designated into four haplogroups (SSA-ECA, SSA-CA, SSA2, and SSA-ESA) using KASP SNP genotyping. SSA-ECA (72%) was the most prevalent and occurred in the northern part of the surveyed area, in the Ituri and Nord/Sud-Kivu provinces, whilst SSA-CA (21%) was present in the south, primarily in Haut-Katanga. SSA-ECA was predominant in the areas of north-eastern DRC most severely affected by cassava brown streak disease and was also reported in the new outbreak area in Pweto territory, Haut-Katanga, in the south. Bemisia afer comprised two major clusters with 85.5% of samples in cluster one, while the rest were in cluster two, which has no reference sequence in GenBank. This study provides important information on the genetic diversity of B. tabaci and B. afer in eastern DRC. This knowledge will be used as a basis for further studies to understand and to identify the role of whitefly haplogroups, their population densities and consequences for virus epidemics and spread as well as leading to improved vector and virus management strategies.

A novel PKC-regulated mechanism controls CD44 ezrin association and directional cell motility.

The dynamic assembly and disassembly of membrane cytoskeleton junctional complexes is critical in cell migration. Here we describe a novel phosphorylation mechanism that regulates the hyaluronan receptor CD44. In resting cells, CD44 is constitutively phosphorylated at a single serine residue, Ser325. After protein kinase C is activated, a switch in phosphorylation results in CD44 being phosphorylated solely at an alternative residue, Ser291. Using fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) and chemotaxis assays we show that phosphorylation of Ser291 modulates the interaction between CD44 and the cytoskeletal linker protein ezrin in vivo, and that this phosphorylation is critical for CD44-dependent directional cell motility.

Phospholipase C-epsilon regulates epidermal morphogenesis in Caenorhabditis elegans.

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP(3))-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP(3) production is stimulated is unknown. IP(3) is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-epsilon produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP(3) receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-gamma and EGL-8/PLC-beta can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-epsilon.

Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision.

Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-delta in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.