Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Genetic Variants in Interleukin-10 Gene Association with Susceptibility and Cervical Cancer Development: A Case Control Study.

Objectives Cervical cancer (CC) is one of the most destructive disease caused by persistent HPV infection which affects women worldwide, especially in developing countries. The genetic basis of host immune response especially cytokine function has been shown to influence CC susceptibility. Studies have demonstrated that IL-10 gene polymorphism have been associated with numerous malignancies, but in context to CC results were inconclusive. Though, aim of our study to investigate the association between IL-10 -1082A/G and -819C/T promoter polymorphism and CC susceptibility. Material and Methods This study comprised 192 women with CC and 200 controls. HPV detection was done by RT-PCR and genotyping was assessed through PCR-RFLP method. Serum concentration of IL-10 measured by ELISA. Results Women with AG and AG+GG genotypes of IL-10 -1082A/G had two-fold increased risk of CC [OR, 2.35 (95% CI, 1.54-3.58), p  = 0.005], [OR, 2.03 (95% CI, 1.36-3.04), p  = 0.0005] compared to controls. Women with G allele of -1082A/G polymorphism had linked with CC susceptibility [OR, 1.39 (95% CI, 1.02-1.88), p  = 0.036] compared to controls. No significant difference was found between patients and controls in the genotype or allele frequencies of IL-10 -819C/T polymorphism [OR, 1.00 (95% CI, 0.63-1.58), p  = 0.99]. The level of serum concentration of IL-10 was significantly higher in cases compared to controls. Conclusion These findings help to understand that polymorphism of IL-10 -1082A/G gene is associated with increased risk of CC development and can serve as a marker of genetic susceptibility to CC.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Identifying strategies to target the metabolic flexibility of tumours.

Plasticity of cancer metabolism can be a major obstacle to efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that, while inhibition of both glutaminase isoforms (Gls1 and Gls2) in tumours considerably delays tumourigenesis, glutamine catabolism continues, owing to the action of amidotransferases. Synergistic inhibition of both glutaminases and compensatory amidotransferases is required to block glutamine catabolism and proliferation of mouse and human tumour cells in vitro and in vivo. Gls1 deletion is also compensated for by glycolysis. Thus, co-inhibition of Gls1 and hexokinase 2 significantly affects Krebs cycle activity and tumour formation. Finally, the inhibition of biosynthesis of either serine (Psat1-KO) or fatty acid (Fasn-KO) is compensated for by uptake of circulating nutrients, and dietary restriction of both serine and glycine or fatty acids synergistically suppresses tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate that either pharmacological or dietary targeting of metabolic compensatory mechanisms can improve therapeutic outcomes.

Association of DNA Repair Genes XRCC1 and APE-1 with the Risk of Cervical Cancer in North Indian population.

BACKGROUNDS: Cervical cancer (CC) is one of the leading cause of death in women worldwide, HPV infection is the major risk factor in the disease development, 0and however other risk factor such as chemical carcinogens, genetic susceptibility and altered immune system are also a cause of the disease progression. In the light of the above statement we studied the base excision repair pathway (BER). METHODS: We identified and studied the association of Single Nucleotide polymorphisms in the DNA repair genes of XRCC1 (Arg194Trp, Arg399G,) and APE-1Asp/148Glu to the susceptibility of cervical cancer (CC) in North Indian population. In our study of cases (n=102). Controls (n=109) were recruited from among women without cervical abnormalities. Genotypes were determined by PCR-CTPP method, Taking DNA from peripheral blood in a case control study. RESULTS: A positive association was observed between the polymorphisms of XRCC1 genes, that is, in codons 194 (P=0.03, odds ratio (OR) =2.39, 95% confidence interval (CI)=5.2-1.1), 280 (P=0.01, OR=4.1, 95% CI=11.5-1.3) and 399 (P=0.01, OR=3.4, 95% CI=8.6-1.3) while APE-1 genotype GG (p=0.03,odds ratio(OR)=0.2,95% confidence interval (CI)=0.97-0.004) we observed a statistically significant protective role in developing cervical cancer. CONCLUSION: Our results suggested that, XRCC1 gene is an important candidate gene for susceptibility to cervical cancer. Although the sample size was small, the present study indicate a statistical association between cervical cancer and XRCC1 SNPs. Future studies are needed that may provide a better understanding of the association between gene polymorphism and cervical carcinoma risk.
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Discovery of a Thiadiazole-Pyridazine-Based Allosteric Glutaminase 1 Inhibitor Series That Demonstrates Oral Bioavailability and Activity in Tumor Xenograft Models.

Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell's ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.

Interfaces between microbes and membranes of host epithelial cells in hemipteran midguts.

Certain families of plant-feeding insects in the order Hemiptera (infraorder Pentatomomorpha) have established symbiotic relationships with microbes that inhabit specific pouches (caeca) of their midgut epithelium. The placement of these caeca in a well-delineated region at the most posterior end of the midgut bordering the hindgut is conserved in these families; in situ the convoluted midgut is predictably folded so that this caecal region lies adjacent to the anterior-most region of the midgut. Depending on the hemipteran family, caeca vary in their number and configuration at a given anterior-posterior location. At the host-microbe interface, epithelial plasma membranes of midgut epithelial cells interact with nonself antigens of microbial surfaces. In the different hemipteran species examined, a continuum of interactions is observed between microbes and host membranes. Bacteria can exist as free living cells within the midgut lumen without contacting host membranes while other host cells physically interact extensively with microbial surfaces by extending numerous processes that interdigitate with microbes; and, in many instances, processes completely envelope the microbes. The host cells can embrace the foreign microbes, completely enveloping each with a single host membrane or sometimes enveloping each with the two additional host membranes of a phagosome.

Luminal membranes in the midgut of the lace bug Corythucha ciliata.

The inordinately long midgut of hemipterans is devoid of peritrophic membranes described for many other insects. These membranes separate apical microvilli of midgut cells from contents of the lumen. In hemipterans, by contrast, contents of the lumen are separated from apical surfaces of midgut epithelia by secretion of additional plasma membranes (perimicrovillar membranes) containing digestive enzymes. In the lace bug Corythucha ciliata, precursors for these perimicrovillar membranes arise in smooth endoplasmic reticula (SER) as stacked, coiled membranes and are continually expelled into the lumen along the entire length of the midgut as stacked, tubular membranes; these membranes undergo changes in form as they pass from the SER to the midgut lumen. Rather than adopting the double membrane configuration in the gut lumen that was first described for hemipteran perimicrovillar membranes, these modified perimicrovillar membranes of the Corythucha gut line apical surfaces of midgut apical lamellae and intermix with the contents of the lumen; foregut and hindgut epithelial cells are devoid of vesicles containing coiled membranes observed abundantly in midgut epithelia. Rather than achieving renewal of adult midgut epithelial cells through the divisions of regenerative cells as observed in many adult insects, prolific generation of perimicrovillar membranes apparently maintains the integrity of this lengthy hemipteran midgut epithelium.

Remodeling of the abdominal epithelial monolayer during the larva-pupa-adult transformation of Manduca.

During metamorphosis of insect epithelial monolayers, cells die, divide, and rearrange. In Drosophila undifferentiated diploid cells destined to form the adult cuticle of each abdominal segment segregate early in development from the surrounding polyploid larval epithelial cells of that segment as eight groups of diploid histoblast cells. The larval polyploid cells are programmed to die and be replaced by divisions and rearrangements of histoblast cells. By contrast, abdominal epithelial cells of Manduca larvae form a monolayer of cells representing different ploidy levels with no definitive segregation of diploid cells destined to form adult structures. These epithelial cells of mixed ploidy levels produce a thick smooth larval cuticle with sparsely distributed sensory bristles. Adult descendants of this larval monolayer produce a thinner cuticle with densely packed scale cells. The transition between these differentiated states of Manduca involves divisions of cells, changes in ploidy levels, and sorting of certain polyploid cells into circular rosette patches to minimize contacts of these polyploid cells with surrounding cells of equal or smaller size. Cells within the rosettes and some surrounding cells are destined to die and be replaced by remaining epithelial cells of uniform size and ploidy at pupa-adult apolysis.

Update in Management of Severe Hypoxemic Respiratory Failure.

Mortality related to severe-moderate and severe ARDS remains high. We searched the literature to update this topic. We defined severe hypoxemic respiratory failure as Pao2/Fio2 < 150 mm Hg (ie, severe-moderate and severe ARDS). For these patients, we support setting the ventilator to a tidal volume of 4 to 8 mL/kg predicted body weight (PBW), with plateau pressure (Pplat) ≤ 30 cm H2O, and initial positive end-expiratory pressure (PEEP) of 10 to 12 cm H2O. To promote alveolar recruitment, we propose increasing PEEP in increments of 2 to 3 cm provided that Pplat remains ≤ 30 cm H2O and driving pressure does not increase. A fluid-restricted strategy is recommended, and nonrespiratory causes of hypoxemia should be considered. For patients who remain hypoxemic after PEEP optimization, neuromuscular blockade and prone positioning should be considered. Profound refractory hypoxemia (Pao2/Fio2 < 80 mm Hg) after PEEP titration is an indication to consider extracorporeal life support. This may necessitate early transfer to a center with expertise in these techniques. Inhaled vasodilators and nontraditional ventilator modes may improve oxygenation, but evidence for improved outcomes is weak.

Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.

Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery.IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery.

LIM kinase inhibitors disrupt mitotic microtubule organization and impair tumor cell proliferation.

The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy.

Discovery, Development, and SAR of Aminothiazoles as LIMK Inhibitors with Cellular Anti-Invasive Properties.

As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.

Regenerative cells and the architecture of beetle midgut epithelia.

The architectural ground plan of beetle and other insect midguts is represented by a monolayer of epithelial cells arranged in a cylindrical configuration. Proliferation and differentiation of regenerative cells maintain the integrity of this monolayer in the face of continual losses of individual cells through cytoplasmic budding and/or expulsion of entire epithelial cells. Peritrophic membranes have conventionally been considered universal features of insect midguts that function to protect vulnerable microvillar surfaces of the midgut epithelium from abrasion by ingested food; however, peritrophic membranes were found in only a small fraction of the adult beetle species examined in this study. In adult beetles, midgut epithelial cells are continually replaced by cells recruited from populations of mitotic regenerative cells that are interspersed among the differentiated epithelial monolayer. To remain contiguous with the other cells in the midgut monolayer, some of these proliferating populations have adopted evaginated configurations of cells that extend for varying distances from the basal surface of the monolayer. These configurations are referred to as regenerative crypts or pouches and consist of progenitor cells and stem cells. The presence, the relative densities, and the relative lengths of these regenerative pouches vary considerably among families of beetles. Placement of longitudinal muscles of the midgut relative to the proximodistal axes of these regenerative pouches also varies among species of beetles. The presence, the size, and the density of regenerative cell populations are related to 1) feeding habits of adult beetles, 2) presence of peritrophic membranes, and 3) expulsion of entire midgut epithelial cells or fragments of these epithelial cells into midgut lumens.

Cell renewal in adjoining intestinal and tracheal epithelia of Manduca.

Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects' responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells.

A novel small-molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vitro and in vivo.

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-kappaB activation; and abrogated the expression of NF-kappaB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 micromol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67-positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (P < 0.05), and abrogated the expression of NF-kappaB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer.

Characterization of the biological effects of a novel protein kinase D inhibitor in endothelial cells.

VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.

Stem cells of the beetle midgut epithelium.

At the completion of metamorphosis, adult insect cells have traditionally been assumed to halt cell divisions and terminally differentiate. While this model of differentiation holds for adult ectodermal epithelia that secrete cuticular specializations of exoskeletons, adult endodermal epithelia are populated by discrete three-dimensional aggregates of stem cells that continue to divide and differentiate after adult emergence. Aggregates of these presumptive adult stem cells are scattered throughout larval and pupal midgut monolayers. At the beginning of adult development (pupal-adult apolysis), the number of cells within each aggregate begins to increase rapidly. Dividing cells form three-dimensional, coherent populations that project as regenerative pouches of stem cells into the hemocoel surrounding the midgut. Stem cell pouches are regularly spaced throughout endodermal monolayers, having adopted a spacing pattern suggesting that each incipient pouch inhibits the formation of a similar pouch within a certain radius of itself-a process referred to as lateral inhibition. At completion of adult development (pupal-adult ecdysis), a distinct basal-luminal polarity has been established within each regenerative pouch. Dividing stem cells occupying the basal region are arranged in three-dimensional aggregates. As these are displaced toward the lumen, they transform into two-dimensional monolayers of differentiated epithelial cells whose apical surfaces are covered by microvilli. This organization of stem cell pouches in insect midguts closely parallels that of regenerative crypts in mammalian intestines.

Neuroglian-positive plasmatocytes of Manduca sexta and the initiation of hemocyte attachment to foreign surfaces.

Observations of hemocyte aggregation on abiotic surfaces suggested that certain plasmatocytes from larvae of Manduca sexta act as foci for hemocyte aggregation. To establish how these particular plasmatocytes form initial attachments to foreign surfaces, they were cultured separately from other selected populations of hemocytes. While all circulating plasmatocytes immunolabel with anti-beta-integrin monoclonal antibody (MAb), only these larger plasmatocytes immunolabel with a MAb to the adhesion protein neuroglian. Neuroglian-negative plasmatocytes and granular cells that have been magnetically segregated from the majority of granular cells adhere to each other but fail to adhere to foreign substrata; by contrast, neuroglian-positive plasmatocytes that segregate with most granular cells adhere firmly to a substratum. Hemocytes form stable aggregates around the large, neuroglian-positive plasmatocytes. However, if neuroglian-positive plasmatocytes are separated from most granular cells, attachment of these plasmatocytes to foreign surfaces is suppressed.