C. elegans is used to demonstrate for the first time the relationship between specific HSPGs and HS modifications in a defined biological process in vivo. HSPGs are critical for the migration of hermaphrodite specific neurons (HSNs) as genetic elimination of multiple HSPGs leads to 80% defect of HSN migration. The effects of genetic elimination of HSPGs are additive, suggesting that multiple HSPGs, present in the migrating neuron and in the matrix, act in parallel to support neuron migration. Genetic analyses suggest that syndecan/sdn-1 and HS 6-O-sulfotransferase, hst-6, function in a linear signaling pathway and glypican/lon-2 and HS 2-O-sulfotransferase, hst-2, function together in a pathway that is parallel to sdn-1 and hst-6. These results suggest core protein specific HS modifications that are critical for HSN migration. In C. elegans, the core protein specificity of distinct HS modifications may be in part regulated at the level of tissue specific expression of genes encoding for HSPGs and HS modifying enzymes. Genetic analysis reveals that there is a delicate balance of HS modifications and eliminating one HS modifying enzyme in a compromised genetic background leads to significant changes in the overall phenotype. These findings are of importance with the view of HS as a critical regulator of cell signaling in normal development and disease.Heparan sulfate proteoglycans (HSPGs) play critical roles in the development and adult physiology of all metazoan organisms. Most of the known molecular interactions of HSPGs are attributed to the structurally highly complex heparan sulfate (HS) glycans. However, whether a specific HSPG (such as syndecan) contains HS modifications that differ from another HSPG (such as glypican) has remained largely unresolved. Here, a neural model in  N-acetylglucosamine (GlcNAc) and glucuronic acid (GluA) residues. The linear HS backbone undergoes a complex pattern of modifications at selective positions. The first modification step is N-deacetylation and subsequent N-sulfation of some glucosamine units. Subsequent epimerization of some GluA units to iduronic acid (IdoA) and sulfation at C2 hydroxyl group of GluA/IdoA and sulfation at C6 or C3 of the glucosamine are actions of a glucuronyl C5-epimerase and heparan 2-, 6- and 3-O-sulfotransferases, respectively. These modifications generally occur in the N-sulfated regions creating domains of high sulfate density (NS-domains), intermediate sulfate density (NS/NA-domains) and low sulfate density (NA-domains). HS biosynthesis is not template-driven and enzymatic reactions do not modify every sugar unit, resulting in huge structural complexity in one of the most heterogeneous molecules in biology.Heparan sulfate (HS) and HS proteoglycans (HSPGs) critically regulate a range of biological processes in metazoan organisms from early events in gastrulation to adult physiology, disease and host response to pathogens  Hs2st leads to complete lack of kidneys and neonatal lethality in vitro, the core protein context is not critical for HS structure determination. Syndecan and glypican from rat embryonic fibroblasts carry HS chains of different molecular mass, but similar charge and structural composition, yet display differential binding to fibronectin, suggesting that the core protein may determine ligand specificity N-sulfation and IdoA units as compared to average tissue HS in vivo.Biochemical analyses have revealed developmental and disease related changes to HS composition. Gene knock-out studies have identified key signaling pathways and developmental processes that require the activities of specific HS biosynthetic enzymes. For example, genetic elimination of mouse C. elegans hermaphrodite specific neurons (HSNs) are a pair of serotonergic neurons that innervate the vulval muscles and are necessary and sufficient to initiate egg laying The C. elegans in vivo. C. elegans has in most cases a single orthologue of vertebrate genes encoding for HSPG core proteins and HS biosynthetic enzymes facilitating analysis without genetic redundancy C. elegans body plan, and were thus chosen as a model to assess HSPGs and HS in neural development. Multiple HSPG dependent neuron guidance pathways are required for HSN migration in parallel. These parallel pathways rely on different HS modifications. Genetic analysis also suggests an interdependence of HS biosynthetic enzymes during HS biosynthesis.This study sought to address the relation between a specific HSPG core protein and distinct HS motifs using a neural development model in Strains- C. elegans strains were maintained at 20°C essentially as described cle-1 (cg120) hse-5 (tm472)rib-2 (tm710) rib-1 (ok556) hst-1 (ok1068); LGX: lon-2 (e678)hst-6 (ok273)sdn-1(zh20)sdn-1(ok449)hst-2(ok595)gpn-1 (ok377) tm734 and tm3006 are deletion alleles of hst-3.1 and hst-3.2, respectively, generated by Shohei Mitani and the Japanese National Bioresource Project. tm734 is a 448 bp deletion and 4 bp insertion eliminating exons 2 and 3 and parts of exon 4 and tm3006 is a 230 bp deletion removing parts of exons two and three. Both mutant alleles were backcrossed at least three times prior to analysis. Both mutants are homozygous viable and fertile and display no overt phenotypic defects as single mutants. The following transgenes were used; zdIs13 [Ptph-1::GFP] mgIs71  sdn-1::gfp transcriptional reporter contains 1.5 kb of sequence upstream from predicted ATG site cloned into SphI/SmaI sites of pPD95.75. The hst-3.1::gfp and hst-3.2::gfp reporters contain a 2 kb of sequence upstream from corresponding predicted ATG site cloned into SphI/XbaI and SphI/BamHI restriction sites of pPD95.75, respectively. DNA constructs were injected at 30 µg/ml. pttx-3::mCherry or ptph-1::mCherry were used as injection markers at 50 µg/ml and pBluescript was used as a filler DNA.Standard molecular biological techniques were used. The C. elegans has two HSNs but sometimes it was not possible to see both HSNs due to the position of the animal. Neuron scores are thus presented as number of total neurons scored, rather than number of animals scored, unless otherwise stated. Statistical significance was tested using either Student’s t-test or Fisher’s test.HSN neuron migration was scored using cell specific fluorescent reporters that are expressed in the HSNs. Each C. elegans was performed by mounting the animals on a 4% agarose pad in a drop of M9 buffer containing 30 mM NaN3 as an anesthetic. HSN migration was scored under fluorescent microscopy using GFP expression in all serotonergic neurons including the HSNs. Compound fluorescent images were acquired using Zeiss AxioCam MRm camera mounted on Zeiss Axioskop2 microscope equipped with x20, x40 and x63 optics. Images were captured using Axiovision. Confocal images were acquired using Leica AOBS SP2 system and images were captured using Leica software. Microscopy images were further cropped and scaled using Adobe Photoshop CS3 .Microscopy of living C. elegans HSPGs more restricted. C. elegans has two orthologues of glypicans, gpn-1lon-2sdn-1cle-1unc-52sdn-1 display the strongest HSN migration defect. A null allele of sdn-1, zh20,ok449 display 52% and 53% HSN migration defects, respectively  and 17%  of all the HSNs failed to migrate at all in sdn-1 mutants (lon-2 (e678) mutants all the HSNs undergo partial migration but 19% (31 out of 163) fail to reach wild type position (gpn-1 (ok377), have wild type HSN migration. Collagen XVIII/cle-1 (cg120) mutants displayed 15% defect in HSN migration  and cle-1 (cg120) enhanced HSN migration defects of sdn-1 (zh20) from 52% (n = 111) to 77%  and 65% (n = 81) respectively  in cle-1 lon-2 sdn-1 triple mutants (lon-2 (e678) and cle-1 (cg120) did not display significantly enhanced HSN migration defect as compared to either single mutant alone . Mutations in gpn-1/glypican did not significantly enhance the HSN migration defect of sdn-1 mutants . However, gpn-1 (ok377) mutation further enhanced the defects seen in lon-2 sdn-1 double mutants from 77% to 84% in triple mutants. Notably, there was a significant increase in the proportion of neurons that failed to migrate at all in lon-2 sdn-1 gpn-1 triple mutants as compared to lon-2 sdn-1 double mutants .To test whether the different HSPGs have redundant functions or whether each HSPG has its own role in HSN migration, HSPG mutants were analyzed in combinations. Mutations in ectively . Similar mutants , suggestzh20 is a null allele of sdn-1/syndecan ok449 is an in-frame deletion of the second and third exons ok449 mutants, but it does not contain any HS chains as detected by monoclonal antibodies against HS  required for the biosynthesis of HS chains rib-1 (ok556) are lethal or sterile rib-2 (tm710) mutants from heterozygous mothers become adults their progeny dies shortly after gastrulation rib-2 (tm710) mutants from heterozygous mothers were analyzed but were found to have no defects in HSN migration . The presence of maternal rib-2 mRNA in the embryo at the time of HSN migration may provide enough RIB-2 protein to mask any defects that result from complete lack of RIB-2. hst-1 encodes an orthologue of ndst, heparan N-de-acetylase-N-sulfotransferase. Deletion mutation in ndst/hst-1 (ok1068) is also homozygous lethal preventing analysis of homozygous offspring from homozygous mutant mothers. Hence the focus on the study was on enzymes modifying HS further in the biosynthetic pathway, mutants of which are all viable. Single mutants in C. elegans heparan C5-epimerase, hse-5 (tm472), and heparan 2-O-sulfotransferases, hst-2 (ok595) have 15% and 30% defects in HSN migration, respectively, whereas single mutants in heparan 6-O-sulfotransferase, hst-6 (ok273), are wild type for HSN migration  are wild type for HSN migration, whereas hst-3.2 (tm3006) mutants have 17% defects in HSN migration  and hst-6 (ok273) significantly enhance the defects in hse-5 epimerase mutants from 15% to 47% and 36% respectively (P<0.001). In contrast, hse-5 hst-2 double mutants are not more severely affected than hst-2 single mutants  HSN migration defect compared to 30% defect in hst-2 single mutants (P<0.001). In hst-2 single mutants only 2% of the HSNs failed to migrate at all and remained positioned in the tail. In contrast, in hst-6 hst-2 double mutants 17% of all HSNs failed to initiate migration suggesting that removing both HS 2- and 6-O-sulfation severely disrupts molecular interactions required for neuron migration. Intriguingly, further removal of one of the 3-O-sulfotransferases, HST-3.1, from hst-6 hst-2 double mutants suppresses the HSN migration defect  of the HSNs are posteriorly misplaced, with only 2% failing to migrate at all. Removal of HST-3.1 in the hst-3.1 hst-2 double mutant also weakly suppresses the HSN migration defects of single hst-2 mutants to 21% . However, genetic elimination of the other 3-O-sulfotransferase, HST-3.2, from hst-2 mutant slightly increases the HSN migration defects to 35% (n = 268) in the hst-3.2 hst-2 double mutant.Genetic elimination of multiple O-sulfotransferases has very robust effects on HSN migration in  mutants ; P<0.001n defect . In hst-Specific HS modifications are critical for defined developmental processes in vertebrates. For example, mice lacking heparan 2-O-sulfation have total renal agenesis hst-3.2 (tm3006) or hst-6 deletion alleles in sdn-1 null background did not alter the severity of the HSN phenotype  as compared to sdn-1 null mutants alone. In contrast, genetic removal of 2-O-sulfates in the sdn-1 null background (sdn-1 hst-2 double mutant) lead to 87% (n = 119) failure of HSN migration to wild type position, with 37% of HSNs failing to migrate at all (hse-5 epimerase and sdn-1 display more severe defects with 61% of HSNs (n = 81) failing to migrate to wild type position and 33% failing to migrate at all . In contrast, additional removal of 2-O-sulfates from glypican/lon-2 and collagen XVIII/cle-1 mutant backgrounds did not significantly alter the neuron defects observed in a single mutant. In lon-2 hst-2 double mutants 37% (n = 91) and in cle-1; hst-2 double mutants 24% of all neurons (n = 129) failed to migrate to wild type position. The pattern of migration defects was not significantly changed either.To gain insight into relationship between HSPG protein cores and specific HS modifications, effects of eliminating HS biosynthetic enzymes in different core protein mutant backgrounds were tested. Eliminating 3- or 6-O-sulfates using e at all . Similarhst-3.1 and hst-3.2 and revealed complementary expression patterns for the two 3-O-sulfotransferases. hst-3.2 reporter is expressed predominantly in neurons  and ok449 (in-frame deletion abolishing HS attachment sites) mutants as detected by monoclonal antibodies recognising the HS “stub” as a result of treatment with heparinase III. Proteins were purified using anion-exchange chromatography (DEAE), which enriches for negatively charged HSPGs. SDN-1 core protein is however present in ok449 mutants as detected by anti-SDN-1 antibodies following immunoprecipitation using anti-SDN-1 antibodies.(TIF)Click here for additional data file.Methods S1HSPG purification and Western blotting. Mixed stage C. elegans were lysed and proteins purified essentially as described (PDF)Click here for additional data file.


TMPRSS2-ERG (T2E) fusion gene and insulin-like growth factor receptor (IGF-1R) to optimize the use of IGF-1R inhibitors.Identifying patients who may benefit from targeted therapy is an urgent clinical issue in prostate cancer (PCa). We investigated the molecular relationship between IGF-1R promoter was evaluated by chromatin immunoprecipitation (ChIP). Sensitivity to anti-IGF-1R agents was evaluated alone or in combination with anti-androgen abiraterone acetate in vitro at basal levels or upon ERG modulation.IGF-1R was analyzed in cell lines and in radical prostatectomy specimens in relation to T2E status. ERG binding to in vitro. ChIP analysis showed that ERG binds IGF-1R promoter and that promoter occupancy is higher in T2E-positive cells. IGF-1R inhibition was more effective in cell lines expressing the fusion gene and combination of IGF-1R inhibitors with abiraterone acetate produced synergistic effects in T2E-expressing cells.IGF-1R analysis performed in PCa cells or clinical samples showed that T2E expression correlated with higher IGF-1R expression at mRNA and protein levels. Genetic modulation of ERG directly affected IGF-1R protein levels Here, we provide the rationale for use of T2E fusion gene to select PCa patients for anti-IGF-1R treatments. The combination of anti-IGF-1R-HAbs with an anti-androgen therapy is strongly advocated for patients expressing T2E. TMPRSS2 and one of the ETS genes, predominantly ERG, was described as being expressed in 40–70% of prostate cancers (PCas) from a radical prostatectomy series [TMPRSS2-ERG (T2E) rearrangement, which is considered an early event because it is found in localized disease more frequently than in high-grade prostatic intraepithelial neoplasia (PIN) [TMPRSS2 contributes only untranslated sequences, the fusion gene results in the overproduction of a truncated ERG protein (tERG) [IGF-1R gene promoter, thus affecting its expression and treatment sensitivity in PCa.Chromosomal translocations are genetic lesions that are produced by illegitimate recombination events between two non-homologous chromosomes or within the same chromosome and that result in chimeric genes . Althougy series . PCa pron (tERG) . ERG shan (tERG) . Studiesn (tERG) , 7, 8. Nn (tERG) . We analn (tERG) , 11. Then (tERG) . Epidemin (tERG) –15. Howen (tERG) , severaln (tERG) , 18. Desn (tERG) , 20. In n (tERG) , 22, putA panel of five prostate cancer cell lines, VCaP, DU-145, PC-3, LNCaP and 22RV1, characterized by different expression levels of the androgen receptor (AR) and T2E gene fusion, and non-malignant RWPE-1 prostate cells  was analIGF-1R gene promoter, and the amount of binding was higher in cells with tERG expression . Because the T2E fusion gene is regulated by androgens, the naturally expressing T2E VCaP cells were treated with abiraterone acetate, and IGF-1R protein levels were investigated upon stimulation. Abiraterone acetate is a second-generation anti-androgen drug that blocks the synthesis of androgens through the inhibition of 17 α-hydroxylase/C17, 20 lyase (CYP17A1). VCaP cells were treated for 72, 96 and 120 h with two concentrations of abiraterone acetate, and western blotting analysis showed that together with a strong ERG down-regulation, IGF-1R levels decreased upon 10 μM treatment in VCaP cells  –26. As sn Figure . Interesn Figure . ConversIGF-1R by qRT-PCR in a retrospective cohort of 270 primary prostate tumors . In particular, patients harboring the fusion gene showed higher IGF-1R mRNA levels, in keeping with the increased binding of ERG to the IGF-1R promoter. This association was confirmed at the protein level. We analyzed the protein expression of ERG and IGF-1R in the same series of patients . As observed at the mRNA level, IGF-1R protein expression was also found to be significantly associated with ERG expression , further verifying the association between IGF-1R and T2E  and were recently authenticated by STR PCR analysis using genRESVR MPX-2 and genRESVR MPX-3 kits (Serac). The following locus were verified: D3S1358, D19S433, D2S1338, D22S1045, D16S539, D18S51, D1S1656, D10S1248, D2S441, TH01, VWA, D21S11, D8S1179, FGA, SE33.Prostate cancer cell lines PC-3, LNCaP, DU-145, VCaP were obtained from the American Type Culture Collection (ATCC). 22RV1 prostate cancer cell line was purchased from Sigma Aldrich. Immortalized non-malignant prostate cell line RWPE-1 and stable trasfectants RWPE-1_tERG or RWPE-1_empty vector were kindly provided by Dr. Gambacorti-Passerini, University of Milano-Bicocca . PC-3, LFundación Instituto Valenciano de Oncología according to the following criteria: specimens obtained from radical retropubic prostatectomies from 1996 to 2002 and no history of previous treatment for PCa (including androgen deprivation therapy or chemotherapy prior to surgery). We identified 270 cases that met these criteria. All patients gave written informed consent for tissue donation for research purposes before tissue collection, and the study was approved by FIVO's Institutional Ethical Committee (ref. number. 2010-19). Clinical data were reviewed from clinical records and stored in a PCa-specific database. Patient characteristics, including the T2E fusion gene status, and demographics are shown in Table in situ hybridization (FISH) as already described [Formalin fixed and paraffin-embedded (FFPE) blocks corresponding to PCa patients were retrieved from the archives of the Biobank of the escribed  and quanIGF-1R) or SYBR Green assays (IR) (Applied Biosystems) as previously reported [IGF-1R (Hs00181385_m1), T2E (Hs03063375_ft) as well as for endogenous control β-2-microglobulin (Hs99999907_m1) were used (Applied Biosystem). Two replicates per gene were considered. Relative quantification analysis was performed on ΔΔCt method [IR and IGF-1R [Cell lines total RNA (2 mg) was extracted with TRIzol (Invitrogen) and purified by precipitation with isopropanol. Oligo dT primers (Applied Biosystems) were used to reverse transcribe RNA. Isolation of RNA from paraffin-embedded tissue was performed using RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion) following providers' specifications and reverse transcription was performed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer's indications. For cell line analysis, Quantitative Real-Time PCR was performed on ABI Prism 7900 (Applied Biosystems) using TaqMan , Pfizer , and Novartis (NVP-AEW541). Abiraterone acetate (S1123) and Cabazitaxel (S3022) were purchased by Selleckchem.TMPRSS2-ERG (isoform 9) and phCMV2 empty vector were kindly provided by Dr. Gambacorti-Passerini, University of Milano-Bicocca [To assess drug sensitivity, MTT assay (Roche) was used according to manufacturer's instructions. Cells were plated into 96 well-plates . After 24 hours, various concentrations of AVE1642 0.01–50 μg/ml), NVP-AEW541(0.03–5 μM), Figitumumab (0.5–500 μg/ml) were added and cells exposed to these drugs for up to 72 hours. phCMV2_HA_tERG plasmid containing the cDNA of the translated sequence of -Bicocca . PC-3 ce0 μg/ml, -Bicocca . siGENOMIGF-1R promoter was evaluated by Real-Time PCR using the following custom TaqMan assay: forward 5′-AGGAGGAGGAGGAGG AGGAG-3′, reverse 5′-GCAGTTCGCAAGATCGCC-3′ and probe 5′-TTGACTCCGCGTTTCTGCCCCTCG-3′. For the TaqMan assay design TFSEARCH - Searching Transcription Factor Binding Sites, version 1.3 free website was used for the prediction of ETS binding sites in the promoter of IGF-1R gene and the sequence spanning from 1041bp to 1051bp was identified as the best. Beacon Designer 4 software was used for the design of the assay spanning from 1005bp to 1114bp. PIM-1 promoter fragment containing ETS consensus sequence was used as immunoprecipitation positive control [ΔCt × input chromatin percentage where ΔCt = Ct (INPUT) - Ct (IP:ERG) [ChIP assay was performed as previously described , 44 usin control  by Real-(IP:ERG) .PCa specimens were incorporated in 11 tissue microarrays (TMA). Two or three representative areas (1 mm in diameter) of each tumor were selected for TMA production by first examining hematoxylin and eosin-stained prostatectomy tumor slides and then sampling tissue from the corresponding paraffin blocks. A tissue microarray instrument (Beecher Instruments) was used for TMA assembly. From TMA blocks, 3-μm-thick sections were immunostained using rabbit anti-human IGF-1Rβ (Santa Cruz Biotechnology) or anti-human ERG clone EP111 polyclonal-Ab (Dako). Percentage of IGF-1R-positive cells and cytoplasmic staining intensity were scored semiquantitatively, forming four groups (from 0 to 3). Cases were scored as low expression when staining intensity was between 0 and 1, and high expression when intensity was 2 and 3.t test. To define drug-drug interactions combination index (CI) was calculated with the isobologram equation [Differences among means where analyzed using two-sided Student's equation  using Ca


T1-mapping using the Shortened Modified Look-Locker Inversion Recovery (ShMOLLI) technique [2 maps were compared. Finally, the feasibility and image quality of systolic T1-mapping (defined as TD=0ms/Mx=176) was tested in 5 patients with tachyarrhythmia .Native T1-maps were acquired using ShMOLLI at 1.5T (Siemens Avanto MR scanner). Healthy volunteers  in normal sinus rhythm (HR 61±9 bpm) provided three short axis  slices to generate an AHA 16-segment model. Serial T1-maps were acquired at varying prescribed trigger delay (TD) times: 0ms, 50ms, 100ms, 150ms, 500ms (MOLLI TD [2=0.996) and systolic readout   with no mistriggering.In patients with tachyarrhythmia  Oxford Biomedical Research Centre based at The Oxford University Hospitals Trust at the University of Oxford. RW acknowledges support from the BHF Centre of Research Excellence, Oxford (RE/08/004).


C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase.Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a  The microtubule (MT) cytoskeleton is required for multiple essential cellular processes including cell division, polarization, and transportation of intracellular cargo. Remodeling of MT structures occurs through changes in MT growth and shrinkage, via the addition or removal of α/β tubulin subunits. The regulation of MT dynamics occurs spatially and temporally as demands on the MT cytoskeleton change throughout the cell cycle. Many proteins interact with MTs to regulate polymer behaviour, for example, the microtubule-associated protein XMAP215 promotes MT polymerization  whereas Xenopus XKCM1 are members of the MT-depolymerizing kinesin-13 family. Unlike conventional kinesins that move along MTs, kinesin-13 proteins utilize energy from ATP hydrolysis to depolymerise MTs -ATP for 30 min at 30°C. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes  and analyzed by autoradiography. Ponceau-S staining was used to confirm that similar amounts of each substrate were added to the reaction  were incubated in kinase buffer . The resulting membrane was blocked with 10% non-fat milk in TBST, and probed with a rabbit anti-KLP-7 polyclonal antibody (1 μg/mL) -ATP incorporation was determined via SDS-PAGE and subsequent autoradiography. Left: AIR-2/ICP-1 phosphorylates GST-KLP-7-N (arrowhead) but not GST-KLP-7-CC (predicted MW of 49 KDa). Middle: The kinase dead AIR-2-KD/ICP-1 does not phosphorylate KLP-7-N nor KLP-7-CC. Right: AIR-2/ICP-1 does not phosphorylate GST alone. AIR-2 auto-phosphorylation is shown by using H2O instead of substrate.AIR-2 kinase and co-activator ICP-1 were incubated with different substrates (TIF)Click here for additional data file.S5 FigThe expression levels of GFP::KLP-7(S546E), GFP::KLP-7(S546A) and GFP::KLP-7(10E) transgenes were determined by western blotting. Transgenic worm lysates were probed with anti-tubulin and anti-KLP-7 antibodies. Seventy young adult hermaphrodites were loaded for each lane.(TIF)Click here for additional data file.S6 Figair-1(RNAi) mitotic embryos were imaged for GFP::KLP-7 fluorescence. The air-1(RNAi) embryos were isolated from worms subjected to RNA-feeding for 14 or 24 hours. Selected time-points are shown; time is relative to the initiation of a mitotic cytokinesis furrow. GFP-KLP-7 levels are reduced in air-1(RNAi) embryos specifically at the centrosomes (arrowheads).Wild-type and (TIF)Click here for additional data file.S7 Figklp-7(tm2143) worms and the maximum velocity of the posterior centrosome during the first 40 seconds of mitotic anaphase was determined. The relevant alteration to the klp-7 transgene is listed. The average (based on n centrosomes) is plotted for GFP-KLP-7(WT), P = 1.0; klp-7(tm2143), P = 0.002; GFP-KLP-7(8A), P = 0.26; GFP::KLP-7(N4E), P = 0.24; GFP::KLP-7(N4A), P = 0.09; GFP::KLP-7(T119E), P = 0.51; GFP::KLP-7(T119A), P = 0.18; GFP::KLP-7(S538ES539E), P = 0.16; GFP::KLP-7(S538AS539A), P = 0.83; GFP::KLP-7(S539E), P = 0.28; GFP::KLP-7(S539A), P = 0.97. klp-7(tm2143) control worms expressed GFP::γ-tubulin and GFP::histone to enable centrosome tracking; all other strains were tracked with the GFP::KLP-7 signal. Error bars are SEM. P-values were based on two-tailed Student’s t-tests comparing each mutant to wild type. ** (0.0005<P<0.005).Different versions of KLP-7-GFP were expressed in (TIF)Click here for additional data file.S8 FigFitted curves (red lines) are shown with the averaged data (blue dots) for each experimental condition. Relevant KLP-7 mutations are indicated.(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.S7 File(XLSX)Click here for additional data file.S8 File(XLSX)Click here for additional data file.S9 File(XLSX)Click here for additional data file.S1 MovieTime (min:sec) is relative to start of MI anaphase.(MOV)Click here for additional data file.S2 MovieTime (min:sec) is relative to start of MI anaphase.(MOV)Click here for additional data file.S3 Movie(MOV)Click here for additional data file.S4 Movie(MOV)Click here for additional data file.S5 Movie(MOV)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Mallory-Denk bodies (MDBs) are hepatocytic protein aggregates found in steatohepatitis and several other chronic liver diseases as well as hepatocellular carcinoma. MDBs are mainly composed of phosphorylated keratins and stress protein p62/Sequestosome-1 (p62), which is a common component of cytoplasmic aggregates in a variety of protein aggregation diseases. In contrast to the well-established role of keratins, the role of p62 in MDB pathogenesis is still elusive. We have generated total and hepatocyte-specific p62 knockout mice, fed them with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce MDBs and allowed the mice to recover from DDC intoxication on a standard diet to investigate the role of p62 in MDB formation and elimination. In the absence of p62, smaller, granular and less distinct MDBs appeared, which failed to mature to larger and compact inclusions. Moreover, p62 deficiency impaired the binding of other proteins such as NBR1 and Hsp25 to MDBs and altered the cellular defense mechanism by downregulation of Nrf2 target genes. Upon recovery from DDC intoxication on a standard diet, there was an enhanced reduction of p62-deficient MDBs, which was accompanied by a pronounced decrease in ubiquitinated proteins. Our data provide strong evidence that keratin aggregation is the initial step in MDB formation in steatohepatitis-related mouse models. Interaction of p62 with keratin aggregates then leads to maturation i.e., enlargement and stabilization of the MDBs as well as recruitment of other MDB-associated proteins. The fate of misfolded proteins gained significant relevance in a variety of diseases characterized by the accumulation of abnormal proteins, collectively known as ‟protein aggregation diseases” , 2. The in vivo. MDBs are hepatocytic protein inclusions found in several chronic liver diseases, especially alcoholic (ASH) or non-alcoholic (NASH) steatohepatitis, idiopathic copper toxicosis, chronic cholestasis, and hepatocellular carcinoma (HCC) . To. Top62-kp62-/- and p62hep-/- mouse strains were included in all experiments. To test for the presence of the Alb-Cre transgene, an additional Cre-specific PCR was performed  for 8 weeks [MDBs were induced in two months old male  8 weeks . To studp62 deletion, reverse transcriptase PCR (RT-PCR) was performed  and immunohistochemical staining’s for K8/K18 and p62 were performed on 4μm thick sections of formaldehyde-fixed paraffin-embedded (FFPE) liver tissue as previously described , 32.Whole liver tissue extracts were prepared using 3% sodium dodecyl sulphate (SDS)-containing buffer. Alternatively, soluble proteins were extracted with Triton-X-100 buffer and keratin-enriched fractions were obtained as described previously taking advantage of their relative insolubility in Triton-X-100 and high-salt buffers . NuclearM120-1 immunofluorescence images and the colocalization efficiency of MM120-1 with other antibodies was carried out with ImageJ interface  using the particle counter by keeping the threshold value equal for all images. Photoshop CS5  was used to prepare the immunofluorescence figures whereas schematics were assembled with CC Illustrator software .The graphs were prepared and statistical analysis was performed using Prism 5 software . All reported p-values are two-tailed and values less than 0.05 were considered statistically significant. Densitometric quantification of protein bands was performed with ImageJ software. Morphometric quantification of Min vivo, we generated p62 total (p62-/-) and hepatocyte-specific (p62hep-/-) knockout mice by Cre-loxP technology M120-1 anM120-1 antibody showed no positivity for cross β-sheet structures in hepatocytes of p62-deficient and corresponding p62f/f and p62help+/+ control mice fed a normal diet  . We obsep62-deficient than in control livers as confirmed by morphometry  ratio in the untreated control, DDC-treated and recovered mouse livers . In chrof/f and p62-/- (f/f livers. This reduced Nrf2 expression was accompanied by a reduction in Nqo1 and Gst mRNA levels in p62-deficient livers when compared to p62f/f livers (Hmox-1 (hemeoxygenase 1) was observed between DDC-intoxicated p62f/f and p62-/- livers  The scheme depicts the non-transgenic (p62NT) allele (upper diagram) consisting of 8 exons (numbered rectangles) and the targeting construct consisting of neomycin resistance selection cassette (NeoR), herpes simplex virus thymidine kinase gene cassette (HSV-TK) and three loxP sequences (represented as block triangles). Two of the loxP sites flank NeoR and the third is located between exon 4 and 5 of the p62 gene. The p62NeoRflox allele was generated by homologous recombination in ES cells. A deletion of NeoR and the first loxP site resulted in the conditional p62f allele while an additional ubiquitous excision of exon 1–4 via crossbreeding with Meu-CRE40 mice yielded the constitutive p62∆ex1-4 or p62-/- knockout. The hepatocyte-specific deletion of exon 1–4 (p62hep-/-) was achieved by breeding p62f mice with animals expressing Cre-recombinase under the control of the liver-specific albumin promoter (Alb-Cre mice). The schemes also include the localization of the genotyping primers LoxPa/b and CreA/B. (B) PCR genotyping of offspring using tail biopsies obtained from the breeding of p62NeoRflox mice with Meu-Cre40 (left panel) and p62f/f mice with Alb-Cre mice (right panel). The alleles and their sizes are shown on the right side of the images. (C) RT-PCR detects the expression levels of p62 in livers and kidney of the highlighted phenotypes. Weak p62 band in the livers of p62hep-/- mice likely corresponds to the signal from non-epithelial cells. β-actin was used as reference for normalization.(TIF)Click here for additional data file.S2 Fig(A) Body weight, (B) liver-to-body weight ratio, (C) serum alanine aminotransferase (ALT) (D) alkaline phosphatase (AP) levels were measured in 8 weeks DDC-fed 4 months old total (p62-/-) and hepatocyte-specific (p62hep-/-) p62-knockout mice as well as in their littermates with unaffected hepatic p62 expression (p62f/f and p62hep+/+). Among DDC-fed mice, p62-/- mice displayed significantly higher body weight than p62f/f animals. DDC induced a substantial increase in liver size and liver enzymes. However, none of these parameters differed between p62-deficient animals and their respective controls. Values are expressed as mean ± SEM. n = 5, *p<0.05.(TIF)Click here for additional data file.S3 Figf/f and p62-/- animals. β-actin was used as reference for normalization. The transcript levels of both K8 and K18 were upregulated in DDC-intoxicated livers of p62f/f and p62-/- mice when compared with untreated mice of both genotypes. Moreover, the increase in transcript expression ratio of K8/K18 was observed in both DDC-intoxicated p62-deficient and wildtype mice when compared with untreated controls. (B) Whole tissue extract from 8 weeks DDC-treated livers of p62f/f and p62-/- mice were immunoblotted for MDB components Hsp70, Hsp25 and Tg2. The expression of Hsp70 did not differ between p62f/f and p62-/- livers. However, the expression of Hsp25 and Tg2 was markedly decreased in p62-/- livers when compared to wildtypes (C) Double immunofluorescence staining with MM120-1 (green) and SMI-31 (red) antibodies was performed on liver sections of DDC-intoxicated p62f/f and p62-/- mice. SMI-31 colocalized with MM120-1 in p62f/f livers wheras no colocalization of SMI-31 was observed in p62-deficient MDBs.(A) qPCR for K8 and K18 was performed on untreated and DDC-treated livers of p62(TIF)Click here for additional data file.S4 FigM120-1 , NBR1 (green) and ubiquitin (red) visualized the distribution of the respective antigens in p62f/f and p62-/- livers intoxicated with DDC for 8 weeks. NBR1-positive and -negative MDBs are highlighted by arrows and dotted circles, respectively. An extensive co-localization of NBR1 and the MDB markers MM120-1 and ubiquitin was seen in animals with intact p62 production (p62f/f and p62hep+/+) but less in the complete (p62-/-) and liver-specific (p62hep-/-) p62-knockouts. .Triple immunofluorescence staining with antibodies against M(TIF)Click here for additional data file.S5 FigM120-1  and desmoplakin  visualized the distribution of the antigens in 8 weeks DDC-intoxicated (8w DDC) and 4 weeks recovered (4w R) p62f/f and p62-/- mouse livers. MDBs are indicated by arrows. .Double immunofluorescence staining with antibodies against M(TIF)Click here for additional data file.S6 Figp62-/- and p62f/f mice of the same treatment regimen.Whole tissue extracts from untreated mice, mice fed with DDC for 8 weeks (8w DDC) and from mice recovered on standard diet for four weeks (4w R) after DDC exposure were analyzed by western blotting with an antibody against LC3 as a marker of autophagy activation. Densitometric analysis depicted the intensity of both LC3-I and -II isoforms (dark/light bar) normalized to β-actin. DDC-intoxicated mice showed. higher LC3-II/I ratio but recovered mice acquired attenuation of autophagy (i.e. lower LC3-II/LC3-I ratio). The extent of autophagy did not differ between total (TIF)Click here for additional data file.S1 TablePCR A/B was performed to distinguish between p62 non-transgenic (p62NT), p62 floxed (p62f) and p62 ∆exon1-4 (p62- or p62hep-) while PCR C was performed to detect the presence of Cre-recombinase.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


PD-L1 is an immune checkpoint molecule expressed by a variety of tumors, including ovarian, which binds to circulating PD-1 expressing effector T cells allowing for tumor escape from the immune system. PD-L1 blockade prevents PD-L1/PD-1 interaction and is currently explored as therapy of solid tumors. Ovarian cancer patients receive combination cisplatin/taxane chemotherapy as standard of care. Chemo-induced effects on tumor PD-L1 expression have been only partially addressed. We studied here the effect of platinum/taxane exposure on PD-L1 expression in vitro and in vivo.Human (OVCA 420 and OVCA432) and mouse (2F8) ovarian cancer cell lines were exposed to increasing doses of cisplatin and paclitaxel for different time periods. PD-L1 expression was analyzed with flow cytometry and Western blot. Through continuous exposure in vitro of mouse 2F8 ovarian cancer cells to increasing doses of cisplatin we have derived a new cisplatin-resistant line (2F8-Cis). In vivo, we have challenged n=37 mice IP with 0.8 million 2F8 cells. Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs, or isotype control every two weeks for three doses starting at day 14 post-inoculation. Tumor- and ascites-derived cancer cells were analyzed with flow cytometry.Exposure of OVCA420 and OVCA432 to cytotoxic doses of cisplatin or paclitaxel trigger PD-L1 up-regulation. Similarly, 2F8-Cis cells show increased cell surface PD-L1 compared to parental 2F8 cells, providing the rationale for combination therapy with PD-L1 blockade. In vivo treatment of mice with aggressive 2F8 tumors respond well to cisplatin and anti-PD-L1 individually with increased survival . At necropsy, anti-PD-L1 therapy significantly reduced tumor burden . Tumor cells cultured from cisplatin-only treated mice expressed higher levels of PD-L1, in line with our in vitro results. A higher percentage of PD-1 expressing cells were found amongst the tumor cells in these cultures versus cisplatin/anti-PD-L1 treated mice. Although high dose anti-PD-L1 immediately following cisplatin administration can control tumor burden (0.48 g), it does not significantly prolong survival (median 29 days). We are currently testing an alternative therapeutic schema exploring a lower anti-PD-L1 dose and a different timing post-chemo.Tumor cells upregulate PD-L1 in response to chemotherapy exposure and combination PD-L1 blockade in conjunction with chemotherapy effectively controls tumor burden. Optimization of timing and dosage for this combination therapy will likely increase its therapeutic benefit.


Cytotoxic anti-neoplastic potency of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] between gemcitabine-equivalent concentrations of 10−12 M and 10−6 M was determined utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKRr-3). The organoselenium compound, [Se]-methylselenocysteine was evaluated to determine if it complemented the anti-neoplastic potency of the covalent gemcitabine immunochemotherapeutics. Results: Gemcitabine-(C4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] and the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] all had anti-neoplastic cytotoxic potency against mammary adenocarcinoma. Gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] produced progressive increases in anti-neoplastic cytotoxicity that were greatest between gemcitabine-equivalent concentrations of 10−9 M and 10−6 M. Dual simultaneous combinations of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] produced levels of anti-neoplastic cytotoxicity intermediate between each of the individual covalent gemcitabine immunochemotherapeutics. Total anti-neoplastic cytotoxicity of the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) was substantially higher when formulated with [Se]-methylsele-nocysteine.The anti-metabolite chemotherapeutic, gemcitabine is relatively effective for a spectrum of neoplastic conditions that include various forms of leukemia and adenocarcinoma/carcinoma. Rapid systemic deamination of gemcitabine accounts for a brief plasma half-life but its sustained administration is often curtailed by sequelae and chemotherapeutic-resistance. A molecular strategy that diminishes these limitations is the molecular design and synthetic production of covalent gemcitabine immunochemotherapeutics that possess properties of selective “targeted” delivery. The simultaneous dual selective “targeted” delivery of gemcitabine at two separate sites on the external surface membrane of a single cancer cell types represents a therapeutic approach that can increase cytosol chemotherapeutic deposition; prolong chemotherapeutic plasma half-life (reduces administration frequency); minimize innocent exposure of normal tissues and healthy organ systems; and ultimately enhance more rapid and thorough resolution of neoplastic cell populations. Materials and Methods: A light-reactive gemcitabine intermediate synthesized utilizing succinimidyl 4,4-azipentanoate was covalently bound to anti-EGFR or anti-HER2/ Al. Alneu (tumumab) –15]..neu (e.gneu) by chemotherapeutic resistant mammary adenocarcinoma (SKBr-3) [4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) applied simultaneously as a dual selectively “targeted” chemotherapeutic regimen. The strategy has clinical relevance in part due to the effectiveness of gemcitabine, especially in combination with paclitaxel, carboplatin and cisplatin following anthracycline failure in the treatment of metastatic breast cancer  and gemcitabine-(C4-amide)-[anti-EGFR] immunochemotherapeutics following separation by column chromatography was determined by measuring absorbance at 265 – 268 nm [Quantitation of the amount of non-covalently bound gemcitabine contained within covalent gemcitabine-(C– 268 nm  21] for for4-ami– 268 nm –24]..4-amide)4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] immunochemotherapeutics was determined by measuring absorbance at 280 nm in combinations with utilizing a 235 nm-vs-280 nm standardized reference curve in order to accommodate for any potential absorption profile over-lap at 280 nm between immunoglobulin and the gemcitabine moiety.Determination of the immunoglobulin concentration for covalent gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] immunochemotherapeutics in addition to reference control anti-EGFR and anti-HER2/neu immunoglobulin fractions were adjusted to a standardized protein concentration of 60 μg/ml and then combined 50/50 v/v with conventional SDS-PAGE sample preparation buffer (Tris/glycerol/bromphenyl blue/SDS) formulated without 2-mercaptoethanol or boiling. Each covalent immunochemotherapeutic, the reference control immunoglobulin fraction (0.9 μg/well) and a mixture of pre-stained reference control molecular weight markers were then developed by non-reducing SDS-PAGE (11% acrylamide) performed at 100 V constant voltage at 3°C for 2.5 hours.Covalent gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] immunochemotherapeutics following mass/size-dependent separation by non-reducing SDS-PAGE were equilibrated in tank buffer devoid of methanol. Mass/size-separated gemcitabine-[anti-HER2/neu] immunochemotherapeutics contained within acrylamide SDS-PAGE gels were then transferred laterally onto sheets of nitrocellulose membrane at 20 volts (constant voltage) for 16 hours at 2°C to 3°C (Note: n = 2 locations) with the transfer manifold packed in crushed ice.Covalent gemcitabine- at 4°C for 15 minutes followed by incubation in TBS blocking buffer solution  containing bovine serum albumin (5%) for 16 hours at 2°C to 3°C applied in combination with gentle horizontal agitation. Prior to further processing, nitrocellulose membranes were vigorously rinsed in Tris buffered saline .Rinsed BSA-blocked nitrocellulose membranes developed for Western-blot (immunodetection) analyses were incubated with biotinylated goat anti-murine IgG  at 4°C for 18 hours applied in combination with gentle horizontal agitation. Nitrocellulose membranes were then vigorously rinsed in TBS  followed by incubation in blocking buffer . Blocking buffer was decanted from nitrocellulose membrane blots which were then rinsed in TBS  before incubation with HRPO-strepavidin  at 4°C for 2 hours applied in combination with gentle horizontal agitation. Prior to chemiluminescent development nitrocellulose membranes were vigorously rinsed in Tris buffered saline . Following development with conjugated HRPO-strepavidin the nitrocellulose membranes were then incubated with HRPO chemiluminescent substrate . Chemiluminescent autoradiography images were acquired by exposing radiographic film (Kodak BioMax XAR) to nitrocellulose membranes sealed within transparent ultra-clear re-sealable plastic bags.ex-vivo model for neoplastic disease. Populations of the mammary adenocarcinoma (SKBr-3) were propagated at ≥85% level of confluency in 150-cc2 tissue culture flasks containing McCoy’s 5a Modified Medium supplemented with fetal bovine serum (10% v/v) and penicillin-streptomycin at a temperature of 37°C under a gas atmosphere of air (95%) and carbon dioxide (5% CO2). Trypsin or any other biochemically active enzyme fraction were not used to facilitate harvest of mammary adenocarcinoma SKBr-3 cell suspensions for seeding of tissue culture flasks or multi-well tissue culture plates. Growth media was not supplemented with growth factors, growth hormones or any other type of growth stimulant.The human mammary adenocarcinoma (SKBr-3) was utilized as an 5/cell), and high over-expression of epidermal growth factor receptor 2 .Characteristic features and biological properties of the mammary adenocarcinoma (SKBr-3) cell line includes chemotherapeutic-resistance, over-expression of epidermal growth factor receptor 1  with PBS followed by their stabilization onto the plastic surface of 96-well plates with paraformaldehyde . Stabilized cellular monolayers were then incubated with covalent gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] immunochemotherapeutics formulated at gradient concentrations of 0.1, 0.25, 0.5, 1.0, 5.0 and 10 μg/ml in tissue culture growth media (200 μl/well). Direct contact incubation between (SKBr-3) cellular monolayers and gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] was performed at 37°C during an incubation period of 3-hours under a gas atmosphere of air (95%) and carbon dioxide (5% CO2). Following serial rinsing with PBS (n = 3), development of stabilized mammary adenocarcinoma (SKBr-3) monolayers entailed incubation with β-galactosidase conjugated goat anti-mouse IgG (1:500 dilution) for 2 hours at 25°C with residual unbound immunoglobulin removed by serial rinsing with PBS (n = 3). Final cell ELISA development required serial rinsing (n = 3) of stabilized (SKBr-3) monolayers with PBS followed by incubation with nitrophenyl-β-D-galactopyranoside substrate . Absorbance within each individual well was measured at 410 nm (630 nm reference wavelength) after incubation at 37°C for a period of 15 minutes.Cell suspensions of mammary adenocarcinoma (SKBr-3) were seeded into 96-well microtiter plates in aliquots of 2 × 104-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] were formulated in growth media at standardized gemcitabine-equivalent concentrations of 10−10, 10−9, 10−8, 10−7, and 10−6 M . Each gemcitabine-equivalent concentration of the covalent immunochemotherapeutics were then transferred in triplicate into 96-well microtiter plates containing mammary adenocarcinoma (SKBr-3) monolayers and growth media (200 μl/well). Covalent immunochemotherapeutics where then incubated in direct contact with monolayer mammary adenocarcinoma (SKBr-3) populations for a period of 182-hours (37°C under a gas atmosphere of air (95%) and carbon dioxide/CO2 (5%). Following the initial 96-hour incubation period, mammary adenocarcinoma (SKBr-3) populations were replenished with fresh tissue culture media with or without covalent gemcitabine-immunochemotherapeutics or benzimidazole tubulin/microtubule inhibitors.Individual preparations of gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] were measured by removing all contents within the 96-well microtiter plates manually by pipette followed by serial rinsing of monolayers (n = 3) with PBS followed by incubation with 3--2,5-diphenyl tetrazolium bromide vitality stain reagent formulated in RPMI-1640 growth media devoid of pH indicator or bovine fetal calf serum (MTT: 5 mg/ml). During an incubation period of 3 – 4 hours at 37°C under a gas atmosphere of air (95%) and carbon dioxide (5% CO2) the enzyme mitochondrial succinate dehydrogenase was allowed to convert the MTT vitality stain reagent to navy-blue formazone crystals within the cytosol of mammary adenocarcinoma (SKBr-3) populations . Contents were then removed from each of the 96-wells in the microtiter plate, followed by serial rinsing with PBS (n = 3). The resulting blue intracellular formazone crystals were dissolved with DMSO (300 μl/well) and then spectrophotometric absorbance of the resulting blue-colored supernatant measured at 570 nm using a computer-integrated microtiter plate reader.Anti-neoplastic cytotoxicity for gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] by microscale desalting/buffer exchange column chromatography consistently yields covalent immunochemotherapeutic preparations that contained <4.0% of residual chemotherapeutic that was not covalently bound to immunoglobulin [4-amide)-[IgG] immunochemotherapeutics was 2.78 utilizing the organic chemistry reaction scheme to form an amide bond at the C4 cytosine-like monoamine of gemcitabine and synthesis of the UV-photoactivated gemcitabine-(C4-amide) intermediate (4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] was relatively larger than the 1.1-to-1 gemcitabine molar-incorporation-index attained during the synthesis of gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu] [Size-separation of gemcitabine--[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] immunochemotherapeutics by SDS-PAGE in combination with immunodetection analysis (Western blot) and chemiluminescent autoradiography recognized a single primary condensed band of 150-kDa between a molecular weight range of 5.0-kDa to 450-kDa (4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] directly corresponds with the known molecular weight/mass of reference control anti-HER2/neu monoclonal immunoglobulin fractions -[anti-HER2/neu] or gemcitabine-(C4-amide)-[anti-EGFR] on the external surface membrane of adherent mammary adenocarcinoma (SKBr-3) populations was measured by cell-ELISA (4-amide)-[anti-HER2/neu] was detected with progressive increases in standardized total immunoglobulin-equivalent concentrations formulated at 0.010, 0.025, 0.050, 0.250, and 0.500 μg/ml (4-amide)-[anti-HER2/neu] or gemcitabine-(C4-amide)-[anti-EGFR] standardized total immunoglobulin-equivalent concentrations had to alternatively be formulated at 0.5, 1.0, 5.0 and 10.0 μg/ml (4-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] for external membrane HER2/neu receptor sites highly over-expressed at 1 × 106/cell on the exterior surface membrane of mammary adenocarcinoma (SKBr-3) populations −6 M (4-amide)-[anti-EGFR] produced measurably higher levels of canti-neoplastic cytotoxicity when incubated with chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) for direct contact periods of 182-hours compared to 96-hours -[anti-EGFR] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) was consistently greater with incubation periods of 182-hours compared to 96-hours (4-amide)-[anti-EGFR] produced progressive anti-neoplastic cytotoxicity that increased with elevations in covalent immunochemotherapeutic at and between the gemcitabine-equivalent concentrations of 10−10 M and 10−6 M for incubation periods of 96-hours and 182-hours respectively (−9 M-to-10−7 M and 10−10 M-to-10−7 M at 96 hours and 182-hours respectively (4-amide)-[anti-EGFR] at 10−6 M for incubation periods of 96-hours and 182-hours respectively (Anti-neoplastic cytotoxicity of gemcitabine-(C96-hours . Gemcitaectively . The mosectively . Maximumectively .4-amide)-[anti-EGFR] produced greater anti-neoplastic cytotoxicity than either gemcitabine (C5-methylcarbamate)-[anti-HER2/neu] [4-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) during an 182-hour incubation period (4-amide)-[anti-HER2/neu] and gemcitabine (C5-methylcarbamate)-[anti-HER2/neu] anti-neoplastic cytotoxicity levels of 14.4% and 9.6%  compared to 63.9%  for gemcitabine-(C4-amide)-[anti-EGFR] at the gemcitabine-equivalent concentration of 10−8 M (4-amide)-[anti-HER2/neu] and gemcitabine (C5-methylcarbamate)-[anti-HER2/neu] both produced progressive but modest increases in anti-neoplastic cytotoxicity that most rapidly increased at and between 10−8 M  and 10−6 M . In contrast, gemcitabine-(C4-amide)-[anti-EGFR] produced anti-neoplastic cytotoxicity levels of 63.9%, 99.3% and 99.6%  at gemcitabine-equivalent concentrations of 10−8 M, 10−7 M and 10−6 M respectively (4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine (C5-methylcarbamate)-[anti-HER2/neu] were 99.6%, 41.1% and 30.8%  at the gemcitabine-equivalent concentration of 10−6 M respectively (Gemcitabine-(CER2/neu]  or gemcin period . Gemcitaf 10−8 M . Gemcitaectively . Maximumectively .4-amide)-[anti-EGFR] compared to gemcitabine alone produced higher levels of anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) at the gemcitabine- equivalent concentrations of 10−10 M , 10−9 M , and 10−8 M  respectively (4-amide)-[anti-EGFR] and gemcitabine at the gemcitabine-equivalent concentrations of 10−7 M and 10−6 M respectively (4-amide)-[anti-HER2/neu] and gemcitabine had nearly identical anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) at gemcitabine-equivalent concentrations of 10−10 M and 10−9 M but gemcitabine was much more potent at 10−8 M , 10−9 M , and 10−6 M  respectively (Gemcitabine-(Cectively  Nearly iectively . Gemcitaectively .4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] evaluated as a gemcitabine-standardized 50/50 molar equivalent formulation in addition to gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] all produced progressive increases in anti-neoplastic cytotoxicity against chemotherapeutic resistant mammary adenocarcinoma (SKBr-3) as a function of increases in gemcitabine-equivalent concentration at and between 10−10 M and 10−6 M (4-amide)-[anti-EGFR] in dual simultaneous combination with gemcitabine-(C4-amide)-[anti-HER2/neu] was 24.3%, 24.5%, 32.8%, 69.9%. and 83.7%  at the gemcitabine-equivalent concentrations of 10−10 M, 10−9 M, 10−8 M, 10−7 M and 10−6 M respectively (4-amide)-[anti-EGFR] in dual simultaneous combination with gemcitabine-(C4-amide)-[anti-HER2/neu] produced levels of anti-neoplastic cytotoxicity that were less than for gemcitabine-(C4-amide)-[anti-EGFR] but consistently greater than levels for gemcitabine-(C4-amide)-[anti-HER2/neu] at and between the gemcitabine-equivalent concentrations of 10−10 M and 10−6 M respectively (4-amide)-[anti-EGFR] in dual simultaneous combination with gemcitabine-(C4-amide)-[anti-HER2/neu] compared to gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] reached maximum or near maximum levels of 83.7%, 99.6%, and 30.8%  respectively -[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] compared to only the dual simultaneous combination of the two covalent gemcitabine immunochemotherapeutics (4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] produced progressive increases in anti-neoplastic cytotoxicity that were most rapid at and between the gemcitabine-equivalent concentrations of 10−9 M and 10−6 M , 93.9% at 10−9 M , 94.7% at 10−8 M , 94.2% at 10−7 M , and 94.2% at 10−6 M  following a direct-contact incubation period  group of the carbohydrate-like moiety of doxorubicin, daunorubicin, epirubicin and other related agents in this class of chemotherapeutics  eit eit44] eapeutics  45]–[56–[56α-moneuticals  [44] ; 4-; 4-N--[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] directly facilitates their binding-avidity for trophic membrane receptor sites  that in turn affords several properties which significantly contribute to the total anti-neoplastic cytotoxic potency of these covalent immunochemotherapeutics. Monoclonal immunoglobulin selected for the synthesis of covalent immunochemotherapeutics should ideally possess several distinct properties that include selective binding-avidity for specific antigenic “sites” on the external surface membrane of cancer cells that are themselves uniquely or highly over-expressed compared to normal, healthy tissues and organ systems. Utilizing immunoglobulin fractions that possess these characteristics allows them to effectively function as a molecular platform that can facilitate selective “targeted” chemotherapeutic delivery in addition to the potential capacity to promote progressive and continual membrane deposition of the chemotherapeutic moiety. The chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) cell type over-expresses EGFR (2.2 × 105/cell) and highly over-expresses HER2/neu (1 × 106/cell) on its exterior surface membrane which promotes selectively “targeted” delivery and progressive membrane deposition of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] at two different endogenous trophic membrane receptor sites. Progressive membrane deposition of gemcitabine-(C4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] or any other analogous covalent immunochemotherapeutic continues as long as sufficient covalent immunochemotherapeutic is present and EGFR and HER2/neu are expressed and re-expressed on the exterior surface membrane. Given this perspective, one of the most critically important mathematical variables related to cancer cell biology that can significantly determine the anti-neoplastic cytotoxicity of covalent immunochemotherapeutics like gemcitabine-[anti-HER2/neu], [neu] [Covalent immunochemotherapeutics can be synthesized that promote selective “targeted” chemotherapeutic delivery in a manner that evoke greater levels of anti-neoplastic cytotoxic potency than the corresponding non-covalent “c moiety  18] [25 [25free”c moiety –68]. Se. Sefree”R2/neu],  24] gem gemfree”], [neu]  [18] [25], [neu]  is the eneu], [neu], [4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu]. Importance of this consideration is based on the realization that in general, it is a prerequisite for most classical chemotherapeutic agents like gemcitabine that possess mechanisms-of-action that is dependent upon enter into the cytosol or nuclear environments in order to create a biological effect. Such processes are assumed to not be a requirement for anti-cancer therapeutics that are membrane-active agents or radioimmunopharmaceuticals that have mechanisms-of-action that do not require entry into cytosol or nuclear environments (e.g. [213Bi or 211At or 224Ra]-anti-TAG-72 for colon carcinoma).In direct accord with the inter-dependent relationship between the immunoglobulin component of covalent immunochemotherapeutics and the biological characteristics of neoplastic cell types, there are other variables in addition to the expression density of membrane-associated “target” sites that significantly determine the anti-neoplastic cytotoxicity of gemcitabine-[anti-HER2/neu],  24] gem gemneu],, [neu],  18] [25 [25neu],, [neu],  and simi, [neu],  have beeneu receptor-mediated endocytosis in populations of mammary adenocarcinoma (SKBr-3) is somewhat limited, other neoplastic cell types like metastatic multiple myeloma are known to internalize and metabolize approximately 8 × 106 molecules of anti-CD74 monoclonal antibody per day [i] selective “targeted” delivery and physical binding at over-expressed and highly over-expressed endogenous receptor sites ; [ii] continual and progressive membrane deposition; [iii] initiation of receptor-mediated endocytosis; and [iv] re-expression/replenishment of uniquely or highly over-expressed endogenous receptors is the potential for gemcitabine-(C4-amide)-[anti-EGFR] and gem-citabine-(C4-amide)-[anti-HER2/neu] and analogous covalent immunochemotherapeutics to promote chemothe-rapeutic moiety accumulation within the cytosol. The degree of cytosol accumulation can approach concentrations that are 8.5× [4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu], gemcitabine-EGF and analogous covalent biochemotherapeutics. Conservative speculation suggests that dual-combinations of covalent immunochemotherapeutics like gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] promote greater levels of simultaneous selective “targeted” gemcitabine delivery/membrane deposition and intracellular gemcitabine internalization at both EGFR and HER2/neu endogenous receptors than can be achieved through selective “targeted” gemcitabine delivery at only a single endogenous membrane receptor over-expressed on the exterior surface membrane of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). The promotion of relatively high cytosol chemotherapeutic concentrations within a short confined time period at least in theory decreases the opportunity and frequency that neoplastic cell sub-populations can develop certain forms of (acquired) chemotherapeutic-resistance.Uniquely or over-expressed endogenous receptor types known to be actively internalized by mechanisms of receptor-mediated endocytosis in response to physical binding of immunoglobulin or receptor ligands represents one of the more preferred type of sites on exterior surface membrane of neoplastic populations that can be utilized to selectively “target” chemotherapeutic delivery while also potentially facilitating profound cytosol chemotherapeutic moiety accumulation  in addit per day . In thisare 8.5×  to 100× are 8.5×  fold greare 8.5× . The deg4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] chemotherapeutic-resistant neoplastic cell types can be attributed to physical properties associated with the relatively large molecular weight of selective delivery platforms that chemotherapeutic moieties are often covalently bound to . Covalent bonding of chemotherapeutics to molecular delivery platforms of relatively large molecular weight effectively prolongs the intravascular pharmacokinetic profile of chemotherapeutic moieties in part because they are no longer removed as rapidly or as extensively from the plasma compartment by renal glomerular filtration (MWCO = 50 – 60 kDa) and excreted into the urine. Furthermore, the chemotherapeutic moiety of covalent immunochemotherapeutic agents do not distribute as extensively into cell populations residing within normal tissues and healthy organ systems because of the large molecular weight of the selective delivery platform (e.g. IgG = 150-kDa) which prevents simple passive diffusion across intact lipid bilayer membranes. The latter consideration is significant because a significantly large percentage of the total dose for a conventional small molecular weight chemotherapeutics within the intravascular compartment ultimately does passively diffuse across and enter the cytosol environment of cell populations in normal tissues and healthy organ systems.Enhanced levels of anti-neoplastic cytotoxicity that are potentially attainable with a dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] or analogous covalent immunochemotherapeutics also inhibits through mechanisms of steric hinderance, the function of biological entities that can utilize chemotherapeutics moieties as a molecular substrate. Enzymes like cytosine deaminase are not as efficient in biochemically degrading or inactivate gemcitabine when it is a moiety within a covalent immunochemotherapeutic. Presumably, at least some degree steric hinderance phenomenon are also responsible for the observation that the non-selective transmembrane efflux “pump”, P-glycoprotein (MDR-1: multi-drug resistance protein) [4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] could therefore function as a molecular strategy for combating patterns of chemotherapeutic resistance in neoplastic cell types. Fortunately, EGFR and HER2/neu trophic membrane receptors are both over-expressed in several resistant forms of breast cancer [The large molecular weight of the immunoglobulin component of gemcitabine-(Cprotein)  commonlyprotein) –77] is  is 4-amiprotein)  [77]–[80protein)  where thprotein)  81]. Al. Al4-amiprotein) . The antprotein) . The covt cancer –84] whe whe4-amit cancer –90]. Re. Re4-amit cancer  92]..4-amide)neu, IGFR, VEGFR and similar trophic membrane receptors that directly or indirectly regulate proliferation kinetics, metastatic behavior and chemotherapeutic-resistance. Similarly, internalization of EGFR, HER2/neu, IGFR, VEGFR or analogous endogenous trophic membrane receptors by mechanisms of receptor-mediated-endocytosis promotes transient down-regulation, or partial to complete depletion of their expression resulting in declines in membrane density that lead to suppression of neoplastic cell viability and proliferation rate. In order for this phenomenon to occur trophic membrane receptor “sites” must to a variable degree become physically depleted in a manner that is partially due to a deficient rate of re-expression and replenishment to original baseline levels. The rate and extent at which trophic receptor complexes are internalized by immunoglobulin-induced receptor-mediated endocytosis is directly determined by the; [i] availability (quantity and concentration) of covalent immunochemotherapeutics; [ii] expression density of membrane “sites” utilized to facilitate selective “targeted” chemotherapeutic delivery and progressive membrane deposition; and the [iii] corresponding rate and extent that uniquely or highly over-expressed trophic membrane receptors or similar “sites” are re-expressed and replenished on the exterior surface of neoplastic cell populations.Utilization of endogenous trophic membrane receptors that regulate neoplastic cell proliferation and viability as “sites” to facilitate selective “targeted” chemotherapeutic delivery on the exterior surface membrane provides an opportunity to potentially exert cytotoxic properties that are independent of those associated with the chemotherapeutic moiety. Most therapeutic immunoglobulins with binding-avidity for uniquely or highly over-expressed endogenous trophic membrane receptors competitively “block” binding of receptor ligands (e.g. EGF ⇉| IgG::EGFR). Suppression of neoplastic cell growth and vitality is therefore achieved by preventing activation, or inhibit the biological function of EGFR, HER2/4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] or any other analogous covalent immunochemotherapeutics at endogenous trophic membrane receptor sites over-expressed on the exterior surface membrane of neoplastic cell types can in an in-vivo environment provide additional levels of selective anti-neoplastic cytotoxicity. Such enhancements in selective anti-neoplastic cytotoxicity that are difficult to comprehensively detect ex-vivo entail and are dependent upon the recruitment of multiple innate immune responses. Given this perspective, binding of covalent immunochemotherapeutics on the exterior surface membrane of neoplastic cells stimulate or activate; [i] complement C9 mediated cytolysis; [ii] opsonization secondary to immunoglobulin binding at endogenous trophic membrane receptor sites and subsequent formation of IgG/receptor/complement complexes (e.g. induced macrophage phagocytosis); and [iii] antibody-dependent cell-mediated cytotoxicity . Despite the potential for gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] to collectively stimulate complement C9 mediated lysis, ADCC responses and promote IgG/complement facilitated opsonization of neoplastic cells in a manner that attains enhanced levels of selective anti-neoplastic cytotoxicity, it continues to be technically difficult to simultaneously simulate and accurately measure each of these three immune-dependent responses utilizing ex-vivo models for neoplastic disease states.Binding of the immunoglobulin component of gemcitabine-, increase neoplastic cell opsonization (e.g. macrophage Fc receptor dependent binding), or promote stimulation of ADCC (e.g. NK lymphocyte Fc receptor dependent binding).In clinical scenarios were immunoglobulin fractions are utilized to selectively “target” delivery of therapeutic pharmaceuticals or diagnostic imaging agents in nuclear medicine the antibody component can be biochemically modified with enzyme preparations like papain in order to cleave and remove the Fc segment of the IgG molecule. Biochemical modifications of this type minimize non-selective binding of immunochemotherapeutics to Fc receptors expressed by cell types that comprise the RE system (mononuclear phagocytic system) that anatomically reside within the spleen and liver. Unfortunately, such biochemical modifications create a covalent immunochemotherapeutic composed predominantly of only F(ab′)4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)- [anti-HER2/neu] at two different trophic membrane receptors over-expressed (e.g. EGFR) or highly over-expressed (e.g. HER2/neu) on a single neoplastic cell type provides a range of opportunities for achieving greater levels of anti-neoplastic cytotoxicity than is possible with only a single covalent gemcitabine immunochemotherapeutic. Gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with gemcitabine-(C4-amide)-[anti-HER2/neu] therefore can provide heightened planes of anti-neoplastic cytotoxicity through several molecular strategies.Dual selective “targeted” binding of both gemcitabine- [Dual simultaneous inhibition of the biological functions and properties of multiple both endogenous trophic membrane receptors or other sites over-expressed on the exterior surface chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Synergistic levels of anti-neoplastic cytotoxicity achieved solely through inhibition of multiple endogenous trophic membrane receptors or analogous biological “targets” can only theoretically be achieved if each different site has distinctly different biological functions/properties within a given neoplastic cell type (e.g. EGFR-vs-CD74)  94]..vs-HER2/neu) [neu applied in simultaneous combination with cyclophosphamide [4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] therefore provides two potential avenues for achieving additive and synergistic levels of cytotoxicity exerted by gemcitabine chemotherapeutic and each anti-trophic receptor immunoglobulin fractions .Selectively “targeted” gemcitabine delivery in dual and simultaneous combination with inhibition of trophic membrane receptor function represents an opportunity for imposing a distinct plane of additive or synergistic anti-neoplastic cytotoxicity especially when trophic receptor “targets” are over-expressed (e.g. SKBr-3: EGFR) or highly over-expressed (e.g. SKBr-3: HER2/neu)  14] [95 [95neu) neu) . Additiv4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] at two different over-expressed trophic receptor types on the exterior surface membrane of a single cancer cell population in-vivo offers the potential to attain a third plane of additive and synergistic anti-neoplastic cytotoxicity from innate immune response mechanisms. Selectively “targeted” additive or synergistic anti-neoplastic cytotoxicity can potentially occur in-vivo through the different combined properties of; [i] complement C9 mediated cytolysis; [ii] IgG/receptor/complement-facilitated opsonization; and [iii] IgG-dependent cell-mediated cytotoxicity (ADCC). Conceptually, at least, the simultaneous binding of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER 2/neu] at two different endogenous trophic receptors on the same cancer cell type offers the probability of evoking a greater degree of selectively “targeted” anti-neoplastic cytotoxicity compared to the selective binding of just a single covalent gemcitabine immunochemotherapeutic.Simultaneous binding of covalent immunochemotherapeutic combinations like gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] in-vivo presents an opportunity to potentially attain still another plane of additive and synergistic anti-neoplastic cytotoxicity that involves; [i] gemcitabine in dual-combination with innate immune responses; [ii] trophic receptor inhibition in dual-combination with innate immune responses; and/or [iii] gemcitabine, trophic receptor inhibition and innate immune responses. In support of this concept, immune cell populations that are involved in ADCC phenomenon release cytotoxic components known to additively and synergistically enhance the cytotoxic anti-neoplastic activity of conventional chemotherapeutic agents [in-vivo compared to levels of anti-neoplastic cytotoxicity observed utilizing ex-vivo based models for neoplastic disease even when the same identical cancer cell types (xenographs) are utilized [4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] collectively serve to explain how the dual-combination of these two covalent immunochemotherapeutics produced additive levels of anti-neoplastic cytotoxicity measured in chemotherapeutic-resistant mammary-adenocarcinoma (SKBr-3) populations functioning as an ex-vivo model for neoplastic disease (4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] were formulated as a 50:50 gemcitabine-equivalent combination the levels of anti-neoplastic cytotoxicity detected were intermediate between each of the two individual gemcitabine immunochemotherapeutics formulated at gemcitabine-equivalent concentrations -[anti-EGFR] in dual simultaneous combination with gemcitabine-(C4-amide)-[anti-HER2/neu].Several variables related to methods and techniques could be have been modified to increase and maximize the anti-neoplastic cytotoxicity of gemcitabine--[anti-EGFR] in dual simultaneous combination with gemcitabine-(C4-amide)-[anti-HER2/neu]. Similarly, human mammary carci-noma (MCF-7/WT-2′) [A human neoplastic cell type other than chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) could have been applied to access anti-neoplastic cytotoxicity of gemcitabine-(C7/WT-2′)  and mamm7/WT-2′)  are both7/WT-2′)  small-ce7/WT-2′)  neurobla7/WT-2′)  or leuke7/WT-2′)  112] be be4-amid7/WT-2′)  61] T-4 T-44-ami7/WT-2′)  [61] cer7/WT-2′)  have bee4-amide)-[anti-EGFR] in dual simultaneous combination with gemcitabine-(C4-amide)-[anti-HER2/neu] would have evoked higher levels of anti-neoplastic cytotoxicity in a different neoplastic cell type specifically sensitive to gemcitabine, the effectiveness of these two covalent gemcitabine immunochemotherapeutics would likely of been higher in neoplastic cell types not displaying profiles of general chemotherapeutic resistance. Majority of the covalent immunochemotherapeutics described in publications to date have measured cytotoxic efficacy utilizing human neoplastic cell populations that are not chemotherapeutic-resistant. Rare exceptions have been the application of chemotherapeutic-resistant metastatic melanoma M21 ;   ) re) re4-ami3H]-thymidine, or an ATP-based assay method would likely have resulted in recognizing lower degrees of early anti-neoplastic cytotoxicity because these analytical modalities reportedly are ≥10-fold more sensitive in detecting lower degrees of early sub-lethal anti-neoplastic cytotoxicity compared to MTT vitality stain based assay methods [ex-vivo In this manner is generally considered to be superior to merely detecting early-stage sub-lethal cellular injury that could potentially be reversible and be more difficult to correlated with in-vivo levels of efficacy and potency.Assessment of neoplastic cellular proliferation with either [ methods  116]. I. I3H]-th methods –61] [11 [113H]-t methods –122]. O. O3H]-th4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] would likely have been greater if their efficacy had been delineated in an in-vivo model for cancer such as human neoplastic xenographs in animal hosts. In such neoplastic disease models, the added effect of host immune responses in the form of selectively “targeted” antibody-dependent cell cytotoxicity (ADCC), complement C9 mediated cytolysis, and/or opsonization/phagocytosis would have been realized.Lastly, as previously eluded to, the anti-neoplastic cytotoxicity of the dual simultaneous combination of the covalent gemcitabine immunochemotherapeutics, gemcitabine-(Cβ-D-arabinofuranosyl-cytosine increases approximately 2.5-fold (e.g. methylseleninate 10 – 100 μM) [Levels of anti-neoplastic cytotoxicity vary between different organoselenium compounds when assessed independently as a single agent or in combination with conventional chemotherapeutics –127]. V. V127].  100 μM) . Synergi 100 μM) . Interes 100 μM) –139] wh whβ-D-ari] induce apoptosis in doxorubicin-resistant lung small-cell carcinoma (selenite 10 μM) [ii] promote severe ER stress (leukemia cell types) [iii] reduce vitality of multidrug-resistant leukemia (selenite-triglycerides 10 μg to 40 μg/ml)  prl types) ; and [ii0 μg/ml) . Several0 μg/ml) . Seleniu0 μg/ml) . Elevate0 μg/ml)  which is2O + O2) . In addi2O + O2) . Seleniu2O + O2)  that occ2O + O2)  and PUMA2O + O2) . Thiored13-imino)-[anti-HER2/neu] [in-vivo administration (4-amide)-[anti-EGFR] in dual simultaneous combination with gemcitabine-(C4-amide)-[anti-HER2/neu] increased substantially in the presence of [Se]-methylselenocysteine formulated at a fixed 15 μM selenium-equivalent concentration (4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] when utilized in concert with [Se]-methylselenocysteine was most prominent at and between the gemcitabine-equivalent concentrations of 10−10 M and 10−7 M but was nearly equivalent at 10−6 M , methylseleninate  126] ha ha126] hER2/neu] . The orgstration . Total antration . Increast 10−6 M . Similarocytosis . Such cl4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] or gemcitabine -[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] each have potent selective “targeted” anti-neoplastic cytotoxic properties against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Applied in dual simultaneous 50/50 combination, gemci-tabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] collectively evoke levels of selective “targeted” anti-neoplastic cytotoxicity that were intermediate between chemotherapeutic-equivalent concentrations of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu]. Vitality/viability profiles for chemotherapeutic-resistant mammary adenocarcinoma were compatible with the concept that gemcitabine was internalized independently but simultaneously at two separate endogenous trophic membrane receptor sites  by mechanisms of receptor-mediated endocytosis. Simultaneous dual selective “targeted” delivery of gemcitabine facilitated by gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] therefore serves as a prototype molecular strategy for maximizing cytosol chemotherapeutic concentrations selectively within a given neoplastic cell type. In this context the anti-EGFR and anti-HER2/neu immunoglobulin components of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] promote [i] selective “targeted” gemcitabine delivery; [ii] progressive gemcitabine deposition on the exterior surface membrane of neoplastic cells; and [iii] accumulation of the gemcitabine moiety within the cytosol to concentrations that are far beyond levels attainable by simple passive diffusion following IV infusion of clinically relevant and safe dosages. While simultaneous selective “targeted” delivery of gemcitabine by gemcitabine-(C4-amide)-[anti-EGFR] applied in dual-combination with gemcitabine-(C4-amide)-[anti-HER2/neu] can potentially improve the resolution of neoplastic disease states, such benefits would in part be made possible through reduction in innocent chemotherapeutic exposure by healthy tissues and normal organ systems.The covalent immunochemotherapeutics, gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu], the anti-neoplastic cytotoxicity attained was substantially greater in the presence of [Se]-methylselenocysteine. Collectively, research investigations with gemcitabine-(C4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] and selenium at least in the form of [Se]-methylselenocysteine demonstrate therapeutic options that may be more effective in resolving chemotherapeutic-resistant neoplastic conditions within a more expedient treatment time frame implementing lower total dosage levels.Selenium in the form of [Se]-methylselenocysteine and methylseleninate both demonstrated anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Applied in concert with the dual simultaneous combination of gemcitabine-(C


Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.C74A mutant.We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4BOur results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control. Dengue disease is a severe health problem in tropical or subtropical areas of the world Basophil/mast cells expressing Fc receptors play an important role in a wide variety of inflammatory reactions and in host defense against pathogens. These cells selectively produce and secrete numerous factors including chemokines, cytokines, lipid mediators, and granule-associated products et al. showed that entry of DENV-antibody complexes into human monocytic cells activated negative regulators DAK and Atg5-Atg12, which then disrupted the RIG-I/MDA-5 signaling cascade and reduced type-1 interferon (IFN) production. This would lead to suppression of IFN-mediated antiviral responses Autophagy is an evolutionarily ancient pathway which contributes to cell survival. Conditions such as nutrient starvation, pathogen infection, and other environmental stresses can induce autophagy. During autophagy, portions of the cytoplasm or small organelles are sequestered into double-membrane vesicles called autophagosomes. Autophagosomes ultimately fuse with lysosomes to generate single-membrane vesicles termed autophagolysosomes, where the contents are subsequently degraded. Transmission electron microscopy (TEM) has been used to detect autophagosome formation, and the expression level of LC3-II is another indicator 2. For the treatment with autophagy inhibitor, cells were pre-incubated with 1 mM 3-methyladenine  for 1 h before DENV infection. 3-MA in 0.5 mM was maintained in the culture medium during the incubation period.Human KU812 basophil precursor cells 2 for 5 days. The cultured medium was harvested and cell debris was removed by centrifugation at 900×g for 10 min. After further centrifugation at 16,000×g for 10 min, the virus supernatant was collected and stored at −80°C until use. Virus titer was determined by plaque assay using the BHK-21 cell line.Dengue virus serotype 2  originally isolated from a Thai patient who suffered from DHF For ADE assay of DENV infection, a dengue-immune serum pool was obtained from nine convalescent-phase sera from patients recovering from DENV2 infection. Dengue-convalescent patient sera were collected in Thailand in 1990 as part of long-standing surveillance and provided by Dr. Bruce Innis  and described previously Aliquots of DENV were resuspended with or without 1∶10,000 dilution of pooled dengue patient sera for 1 h at 4°C. KU812 or HMC-1 cells were incubated with DENV (with or without pooled dengue patient sera) at MOI of 1 for 90 min at 4°C. Cells were then washed twice with RPMI medium to remove unabsorbed virus and antibodies. Cells were resuspended and supplemented with 2% FBS-containing medium at 37°C for further incubation.5 cells/well) and cultured in DMEM under CO2-enriched conditions. Supernatants and cell lysates from DENV-infected cells were serially diluted and inoculated with BHK-21 cells for plaque assay. After 2 h post-infection, the solution was replaced with fresh DMEM containing 2% FBS and 0.5% methyl cellulose (Sigma-Aldrich). At five days post-infection, the medium was removed, and the cells were fixed and stained with 1% crystal violet, 0.64% NaCl, and 2% formalin (Sigma-Aldrich).BHK-21 cells were plated onto 12-well plates  at room temperature for 10 min. Fc receptors of cells were blocked with 1∶100 dilution (in permeabilizing buffer) of normal human sera  at 4°C for 1 h. After washing, cells were then stained with anti-DENV envelope (E) protein or anti-nonstructural protein 4B (NS4B) (GeneTex) at 4°C for 30 min. Cells were incubated with Alexa488-conjugated secondary antibody (Life Technologies) at 4°C for 30 min and analyzed using FACS Calibur (BD Biosciences). For the anti-E antibody-enhanced DENV infection experiment, cells were then stained with FITC-conjugated anti-E antibodies at 4°C for 1 h and analyzed using FACS Calibur. For the Atg4B mutant-transfected antibody-enhanced DENV infection experiment, cells were stained with anti-NS4B antibodies at 4°C for 30 min, followed by Alexa647-conjugated secondary antibody (Life Technologies) at 4°C for 30 min, and analyzed using an LSRFortessa instrument (BD Biosciences).Cells were fixed with 1% formaldehyde (Sigma-Aldrich), permeabilized with 0.1% saponin, and then blocked Fc receptors with normal human sera. Cells were then stained with anti-E, anti-double strand RNA (dsRNA) (English & Scientific Consulting), or anti-LC3 (MBL) antibodies at 4°C for overnight. Cells were then incubated with Alexa488- or Alexa594-conjugated secondary antibody (Life Technologies) at 4°C for 1 h and analyzed with a FV1000 confocal microscope (Olympus).C74A) were gifts of Dr. Tamotsu Yoshimori . Briefly, the Atg4B cDNA was cloned from genomic DNA isolated from mouse embryonic fibroblast (MEF) cells and was inserted into pmStrawberry-C1 using engineered BamHI and KpnI sites; the point mutation was introduced using the QuikChange Site-Directed mutagenesis system (Stratagene) 7 cells) were resuspended in 0.6 ml RPMI-1640 medium containing 10 mg/ml BSA, and mixed with 20 µg plasmid DNA in electroporation cuvette (0.4-cm electrode gap) and subjected to 220 V for 70 msec by Gene Pulser II Electroporation System (Bio-Rad). After electroporation, the cells were washed with fresh medium and then cultured for 48 h before DENV infection.The plasmids . The cells were further dehydrated with ethanol and embedded with LR White. Ultrathin sections were stained with uranyl acetate and lead citrate (Sigma-Aldrich), and then observed using a HT7000 transmission electron microscope (Hitachi).Twenty-four h after DENV infection, KU812 cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in 0.1 M cacodylate buffer containing 4% sucrose, 1 mM MgClFollowing infection with DENV, cells were lysed using a Triton X-100 based lysis buffer with a protease inhibitor mix and phosphatase inhibitors (Sigma-Aldrich). Cell extract was separated using SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). After blocking with 5% nonfat milk in PBS-T (0.05% Tween-20), blots were probed with anti-LC3, -NS4B, -p62 (MBL), and -β-actin (Santa Cruz Biotechnology) antibodies at 4°C for overnight. After washing with PBS-T, blots were treated with horseradish peroxidase (HRP)-conjugated secondary antibodies  in 1∶5000 dilution at room temperature for 1 h. Blots were developed using western lightning chemiluminescence reagent .t-test for statistical analysis. Statistical significance was set at P<0.05.We used the paired Recent reports demonstrated that autophagy is observed in several kinds of DENV-infected cells We further confirmed autophagy in DENV-infected KU812 cells using confocal microscopy to analyze the expression of DENV E protein and LC3 punctation, as a marker for autophagy. In addition to increased DENV E protein expression . The co-We further used purified anti-DENV E protein monoclonal antibody 137-22 (a gift from Dr. Huan-Yao Lei's laboratory) We further confirmed autophagy and DENV infection in the immature mast cell line HMC-1. After 24 h post-infection of DENV alone (MOI = 1), few HMC-1 cells expressed E protein and LC3 punctation. In contrast, antibody-enhanced DENV infection of HMC-1 cells resulted in increased levels of E protein and LC3 punctation . Co-locaC74A-expressing KU812 cells. Atg4BC74A is an inactive Atg4B mutant due to the mutation of the catalytic cysteine residue (Cys74) and in consequence lacks protease activity. Atg4BC74A can hamper autophagosome closure and has been utilized as a useful tool to inhibit autophagy C74A-expressing cells after transfection with Strawberry-Atg4BC74A plasmids in nutrient-rich and starvation conditions (C74A plasmids (transfection efficiency 14.1%), we analyzed the infectivity only from the cells showing red fluorescence. We counted the same numbers of Strawberry- and Strawberry-Atg4BC74A-expressing cells , with DENV alone, or with DENV in the presence of sub-neutralizing dengue patient sera. After 24 h post-infection, cells were fixed, permeabilized, and stained with anti-double strand (ds) RNA (red), LC3 protein (green), and DAPI (blue). Cells were then mounted and observed by confocal microscopy. The filled arrowheads indicate the dsRNA-positive cells, the empty arrowheads indicate the cells with LC-3 punctation, and the arrows indicate the cells in which dsRNA is co-localized with LC3 punctation. The square areas are zoomed-in images and shown in the right panels . Bar: 10 µm. The imaging data were repeated three times and one set of representative results is shown.(TIF)Click here for additional data file.Figure S2The autophagy inhibitor 3-MA reduces DENV infection. KU812 cells were pre-treated with or without 5 mM 3-MA for 1 h before incubation with medium alone (Mock), DENV alone, or DENV with sub-neutralizing dengue patient sera. 3-MA was maintained in the medium during DENV infection. After 24 h post-infection, the expression of DENV E protein and NS4B protein was detected by flow cytometry. A representative histogram of each group is shown.(TIF)Click here for additional data file.Figure S3C74A-expressing KU812 cells.Autophagy is inhibited in the strawberry-Atg4B (A) KU812 cells were transfected with strawberry or strawberry-Atg4BC74A plasmids. After transfection and incubation for 48 h, strawberry- and strawberry-Atg4BC74A-expressing KU812 cells were incubated in the nutrient-rich medium or Hank's balanced salt solution (starvation). After 3 h, cells were fixed, permeabilized, stained, and observed by confocal microscopy. The filled arrowheads indicate the strawberry- and strawberry-Atg4BC74A-expressing cells (red). The empty arrowheads indicate LC3 punctation (green). The arrows indicate the cells which possess both green and red fluorescence. The imaging data were repeated two times and one set of representative results is shown. Bar: 20 µm (B) The percentage of LC3 punctation from red cells was quantified from two independent experiments.(TIF)Click here for additional data file.Figure S4Blockade of LC3 reduces DENV infection. KU812 cells were transfected with shRNA specifically targeting luciferase (shLuc) or LC3 (shLC3). The targeting sequence on luciferase is 5′-GCGCCATTCTATCCGCTGGAA-3′ and the targeting sequence on LC3 is 5′-CGCTTACAGCTCAATGCTAAT-3′. (A) The knockdown efficiency of LC3 in the cells of each group was shown using Western blot analysis. (B) After transfection and incubation for 48 h, cells were infected with DENV alone or DENV with sub-neutralizing dengue patient sera. After 24 h post-infection, the expression of DENV NS4B protein was detected by flow cytometry. A representative histogram of each group is also shown in the right panel.(TIF)Click here for additional data file.