Analysis of Human Milk Microbiota in Northern Greece by Comparative 16S rRNA Sequencing vs. Local Dairy Animals.

Milk is a biological fluid with a dynamic composition of micronutrients and bioactive molecules that serves as a vital nutrient source for infants. Milk composition is affected by multiple factors, including genetics, geographical location, environmental conditions, lactation phase, and maternal nutrition, and plays a key role in dictating its microbiome. This study addresses a less-explored aspect, comparing the microbial communities in human breast milk with those in mature milk from species that are used for milk consumption. Since mature animal milk is used as a supplement for both the infant (formula) and the child/adolescent, our main aim was to identify shared microbial communities in colostrum and mature human milk. Using 16S rRNA metagenomic sequencing, we focused on characterizing the milk microbiota in the Northern Greek population by identifying shared microbial communities across samples and comparing the relative abundance of prevalent genera. We analyzed ten human milk samples (from five mothers), with five collected three days postpartum (colostrum) and five collected thirty to forty days postpartum (mature milk) from corresponding mothers. To perform an interspecies comparison of human milk microbiota, we analyzed five goat and five bovine milk samples from a local dairy industry, collected fifty to seventy days after birth. Alpha diversity analysis indicated moderate diversity and stability in bovine milk, high richness in goat milk, and constrained diversity in breast milk. Beta diversity analysis revealed significant distinctions among mammalian species, emphasizing both presence/absence and abundance-based clustering. Despite noticeable differences, shared microbial components underscore fundamental aspects across all mammalian species, highlighting the presence of a core microbiota predominantly comprising the Proteobacteria, Firmicutes, and Actinobacteriota phyla. At the genus level, Acinetobacter, Gemella, and Sphingobium exhibit significant higher abundance in human milk compared to bovine and goat milk, while Pseudomonas and Atopostipes are more prevalent in animal milk. Our comparative analysis revealed differences and commonalities in the microbial communities of various mammalian milks and unraveled the existence of a common fundamental milk core microbiome. We thus revealed both species-specific and conserved microbial communities in human, bovine, and goat milk. The existence of a common core microbiome with conserved differences between colostrum and mature human milk underscores fundamental similarities in the microbiota of milk across mammalian species, which could offer valuable implications for optimizing the nutritional quality and safety of dairy products as well as supplements for infant health.

Combining SiRPα decoy-coengineered T cells and antibodies augments macrophage-mediated phagocytosis of tumor cells.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five Aspergillus Species.

Aspergillus mold is a ubiquitously found, airborne pathogen that can cause a variety of diseases from mild to life-threatening in severity. Limitations in diagnostic methods combined with anti-fungal resistance render Aspergillus a global emerging pathogen. In industry, Aspergilli produce toxins, such as aflatoxins, which can cause food spoilage and pose public health risk issues. Here, we report a multiplex qPCR method for the detection and identification of the five most common pathogenic Aspergillus species, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans. Our approach exploits species-specific nucleotide polymorphisms within their ITS genomic regions. This novel assay combines multiplex single-color real time qPCR and melting curve analysis and provides a straight-forward, rapid, and cost-effective detection method that can identify five Aspergillus species simultaneously in a single reaction using only six unlabeled primers. Due to their unique fragment lengths, the resulting amplicons are directly linked to certain Aspergillus species like fingerprints, following either electrophoresis or melting curve analysis. Our method is characterized by high analytical sensitivity and specificity, so it may serve as a useful and inexpensive tool for Aspergillus diagnostic applications both in health care and the food industry.

Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris.

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

High-Resolution Melting (HRM) Analysis for Rapid Molecular Identification of Sparidae Species in the Greek Fish Market.

The red porgy (Pagrus pagrus) and the common dentex (Dentex dentex) are Sparidae species of high commercial value, traded in the Greek market. In some cases, fish species identification from Greek fisheries is difficult for the consumer due to the strong morphological similarities with their imported counterparts or closely related species such as Pagrus major, Pagrus caeroleustictus, Dentex gibbosus and Pagellus erythrinus, especially when specimens are frozen, filleted or cooked. Techniques based on DNA sequencing, such as COI barcoding, accurately identify species substitution incidents; however, they are time consuming and expensive. In this study, regions of mtDNA were analyzed with RFLPs, multiplex PCR and HRM in order to develop a rapid method for species identification within the Sparidae family. HRM analysis of a 113 bp region of cytb and/or a 156 bp region of 16s could discriminate raw or cooked samples of P. pagrus and D. dentex from the aforementioned closely related species and P. pagrus specimens sampled in the Mediterranean Sea when compared to those fished in the eastern Atlantic. HRM analysis exhibited high accuracy and repeatability, revealing incidents of mislabeling. Multiple samples can be analyzed within three hours, rendering this method a useful tool in fish fraud monitoring.

Development of Multiplex PCR and Melt-Curve Analysis for the Molecular Identification of Four Species of the Mullidae Family, Available in the Market.

The authentication of food products and the verification of their identity are of major importance for consumers. Food fraud through mislabeling is an illegal practice consisting of the substitution of an expensive food product by a relatively cheaper one, misleading false labelling of their origin and adulteration in processed or frozen products. This issue is particularly of high importance concerning fish and seafood, which are easily adulterated primarily due to difficult morphological identification. Fish species of the Mullidae family are considered among the most high-valued seafood products traded in Greece and Eastern Mediterranean in general, in terms of the price and demand. Specifically, the red mullet (Mullus barbatus) and the striped red mullet (Mullus surmuletus) are both indigenous in the Aegean (FAO Division 37.3.1) and the Ionian (FAO Division 37.2.2) Seas, with high levels of consumers' preferences. However, they could be easily adulterated or misidentified by the invasive Aegean Sea Lessepsian migrator goldband goatfish (Upeneus moluccensis) as well as by the imported West African goatfish (Pseudupeneus prayensis). Keeping this in mind, we designed two novel, time-saving and easy-to-apply multiplex PCR assays and one multiple Melt-Curve analysis real-time PCR for the identification of these four species. These methodologies are based on species-specific primers targeting single nucleotide polymorphisms (SNPs) detected via sequencing analysis of the mitochondrial cytochrome C oxidase subunit I (CO1) and of the cytochrome b (CYTB) genes in newly collected individuals, with additional comparison with congeneric and conspecific haplotypes obtained from the GenBank database. Both methodologies, targeting CO1 or CYTB, utilize one common and four diagnostic primers, producing amplicons of different length that are easily and reliably separated on agarose gel electrophoresis, yielding a single clear band of diagnostic size for each species or a certain Melt-Curve profile. The applicability of this cost-effective and fast methodology was tested in 328 collected specimens, including 10 cooked samples obtained from restaurants. In the vast majority (327 out of the 328) of the specimens tested, one single band was produced, in agreement with the expected products with a single exception a M. barbatus sample that was identified as M. surmuletus, the identity of which was confirmed using sequencing, indicating erroneous morphological identification. The developed methodologies are expected to contribute to the detection of commercial fraud in fish authentication.

TEM1-targeting PEGylated PLGA shikonin nanoformulation for immunomodulation and eradication of ovarian cancer.

Introduction: Tumor endothelial marker 1 (TEM1) is expressed by tumor vascular endothelial cells in various cancers. Methods: Here, we developed poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) PEGylated with polyethylene glycol (PEG) and functionalized with anti-TEM1 antibody fragment (78Fc) and loaded them with necroptosis-inducing agent shikonin (SHK) (78Fc-PLGA-SHK NPs). Results: The nanoformulation showed a smooth spherical shape (~120 nm; the ζ potential of -30 mV) with high drug entrapment and bioconjugation efficiencies (~92% and ~90%, respectively) and a sustained-release profile in serum. Having significant toxicity in vitro (e.g., MS1 and TC1 cells), the nanoformulation dramatically increased the cytotoxicity in the TC1 murine lung carcinoma subcutaneous and intravenous/metastatic models as aggressive tumor models. The injection of the 78Fc-PLGA-SHK NPs to the MS1-xenograft mice resulted in significantly higher accumulation and effects in the TEM1-positive tumor targets, while they were excreted via urine track without retaining in the liver/spleen. In the TC1 subcutaneous model, C57/BL6 mice treated with the 78Fc-PLGA-SHK NPs revealed a significant therapeutic effect. The mice, which were tumor-free after receiving the nanoformulation, were re-challenged with the TC1 cells to investigate the immune response. These animals became tumor-free a week after the injection of TC1 cells. Conclusion: Based on these findings, we propose the 78Fc-PLGA-SHK NPs as a highly effective immunostimulating nanomedicine against the TEM1-expressing cells for targeted therapy of solid tumors including ovarian cancer.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Higher-order immunoglobulin repertoire restrictions in CLL: the illustrative case of stereotyped subsets 2 and 169.

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, ∼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection.

TRIP - T cell receptor/immunoglobulin profiler.

BACKGROUND: Antigen receptors are characterized by an extreme diversity of specificities, which poses major computational and analytical challenges, particularly in the era of high-throughput immunoprofiling by next generation sequencing (NGS). The T cell Receptor/Immunoglobulin Profiler (TRIP) tool offers the opportunity for an in-depth analysis based on the processing of the output files of the IMGT/HighV-Quest tool, a standard in NGS immunoprofiling, through a number of interoperable modules. These provide detailed information about antigen receptor gene rearrangements, including variable (V), diversity (D) and joining (J) gene usage, CDR3 amino acid and nucleotide composition and clonality of both T cell receptors (TR) and B cell receptor immunoglobulins (BcR IG), and characteristics of the somatic hypermutation within the BcR IG genes. TRIP is a web application implemented in R shiny. RESULTS: Two sets of experiments have been performed in order to evaluate the efficiency and performance of the TRIP tool. The first used a number of synthetic datasets, ranging from 250k to 1M sequences, and established the linear response time of the tool (about 6 h for 1M sequences processed through the entire BcR IG data pipeline). The reproducibility of the tool was tested comparing the results produced by the main TRIP workflow with the results from a previous pipeline used on the Galaxy platform. As expected, no significant differences were noted between the two tools; although the preselection process seems to be stricter within the TRIP pipeline, about 0.1% more rearrangements were filtered out, with no impact on the final results. CONCLUSIONS: TRIP is a software framework that provides analytical services on antigen receptor gene sequence data. It is accurate and contains functions for data wrangling, cleaning, analysis and visualization, enabling the user to build a pipeline tailored to their needs. TRIP is publicly available at https://bio.tools/TRIP_-_T-cell_Receptor_Immunoglobulin_Profiler .

The homeodomain transcription factor MEIS1 triggers chemokine expression and is involved in CD8+ T-lymphocyte infiltration in early stage ovarian cancer.

CD8+ T-lymphocytes infiltration is a favorable prognostic marker in ovarian cancer. Recently we identified MEIS1 as a gene overexpressed in early stage ovarian tumors enriched for CD8+ T-cells. Here, we report the molecular mechanism of the homeodomain transcription factor MEIS1 in lymphocyte recruitment. We validated that MEIS1 expression is a positive predictor of CD8+ T cells in early stage ovarian cancer. We showed that MEIS1 induces the expression of CCL18, CCL4, CXCL7, CCL5, CXCL1, and IL8 chemokines in cancer cells followed by their secretion in the culture medium ultimately triggering CD8+ T-lymphocyte recruitment in vitro. Knock down of MEIS1 expression by siRNA resulted in downregulation of these chemokines. We verified that MEIS1 binds to the promoters of chemokine genes, both in vitro and in vivo. We also showed that the expression levels of MEIS1 correlated tightly with the mRNA levels of chemokines CCL4 and CCL18 in early stage ovarian cancer patient samples and served as a positive prognostic marker, as shown by Kaplan-Meyer survival analysis. In conclusion, we propose that MEIS1 plays a pivotal role in the regulatory circuitry governing T-cell chemo-attraction during the early stages of ovarian cancer.

Roles of NF-κB Signaling in the Regulation of miRNAs Impacting on Inflammation in Cancer.

The NF-κB family of transcription factors regulate the expression of genes encoding proteins and microRNAs (miRNA, miR) precursors that may either positively or negatively regulate a variety of biological processes such as cell cycle progression, cell survival, and cell differentiation. The NF-κB-miRNA transcriptional regulatory network has been implicated in the regulation of proinflammatory, immune, and stress-like responses. Gene regulation by miRNAs has emerged as an additional epigenetic mechanism at the post-transcriptional level. The expression of miRNAs can be regulated by specific transcription factors (TFs), including the NF-κB TF family, and vice versa. The interplay between TFs and miRNAs creates positive or negative feedback loops and also regulatory networks, which can control cell fate. In the current review, we discuss the impact of NF-κB-miRNA interplay and feedback loops and networks impacting on inflammation in cancer. We provide several paradigms of specific NF-κB-miRNA networks that can regulate inflammation linked to cancer. For example, the NF-κB-miR-146 and NF-κB-miR-155 networks fine-tune the activity, intensity, and duration of inflammation, while the NF-κB-miR-21 and NF-κB-miR-181b-1 amplifying loops link inflammation to cancer; and p53- or NF-κB-regulated miRNAs interconnect these pathways and may shift the balance to cancer development or tumor suppression. The availability of genomic data may be useful to verify and find novel interactions, and provide a catalogue of 162 miRNAs targeting and 40 miRNAs possibly regulated by NF-κB. We propose that studying active TF-miRNA transcriptional regulatory networks such as NF-κB-miRNA networks in specific cancer types can contribute to our further understanding of the regulatory interplay between inflammation and cancer, and also perhaps lead to the development of pharmacologically novel therapeutic approaches to combat cancer.

A step-by-step microRNA guide to cancer development and metastasis.

BACKGROUND: Cancer is one of the leading causes of mortality. The neoplastic transformation of normal cells to cancer cells is caused by a progressive accumulation of genetic and epigenetic alterations in oncogenes, tumor suppressor genes and epigenetic regulators, providing cells with new properties, collectively known as the hallmarks of cancer. During the process of neoplastic transformation cells progressively acquire novel characteristics such as unlimited growth potential, increased motility and the ability to migrate and invade adjacent tissues, the ability to spread from the tumor of origin to distant sites, and increased resistance to various types of stresses, mostly attributed to the activation of genetic stress-response programs. Accumulating evidence indicates a crucial role of microRNAs (miRNAs or miRs) in the initiation and progression of cancer, acting either as oncogenes (oncomirs) or as tumor suppressors via several molecular mechanisms. MiRNAs comprise a class of small ~22 bp long noncoding RNAs that play a key role in the regulation of gene expression at the post-transcriptional level, acting as negative regulators of mRNA translation and/or stability. MiRNAs are involved in the regulation of a variety of biological processes including cell cycle progression, DNA damage responses and apoptosis, epithelial-to-mesenchymal cell transitions, cell motility and stemness through complex and interactive transcription factor-miRNA regulatory networks. CONCLUSIONS: The impact and the dynamic potential of miRNAs with oncogenic or tumor suppressor properties in each stage of the multistep process of tumorigenesis, and in the adaptation of cancer cells to stress, are discussed. We propose that the balance between oncogenic versus tumor suppressive miRNAs acting within transcription factor-miRNA regulatory networks, influences both the multistage process of neoplastic transformation, whereby normal cells become cancerous, and their stress responses. The role of specific tumor-derived exosomes containing miRNAs and their use as biomarkers in diagnosis and prognosis, and as therapeutic targets, are also discussed.

Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts.

Senescence recapitulates the ageing process at the cell level. A senescent cell stops dividing and exits the cell cycle. MicroRNAs (miRNAs) acting as master regulators of transcription, have been implicated in senescence. In the current study we investigated and compared the expression of miRNAs in young versus senescent human fibroblasts (HDFs), and analysed the role of mRNAs expressed in replicative senescent HFL-1 HDFs. Cell cycle analysis confirmed that HDFs accumulated in G1/S cell cycle phase. Nanostring analysis of isolated miRNAs from young and senescent HFL-1 showed that a distinct set of 15 miRNAs were significantly up-regulated in senescent cells including hsa-let-7d-5p, hsa-let-7e-5p, hsa-miR-23a-3p, hsa-miR-34a-5p, hsa-miR-122-5p, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-503-5p, hsa-miR-574-3p, hsa-miR-574-5p and hsa-miR-4454. Importantly, pathway analysis of miRNA target genes down-regulated during replicative senescence in a public RNA-seq data set revealed a significant high number of genes regulating cell cycle progression, both G1/S and G2/M cell cycle phase transitions and telomere maintenance. The reduced expression of selected miRNA targets, upon replicative and oxidative-stress induced senescence, such as the cell cycle effectors E2F1, CcnE, Cdc6, CcnB1 and Cdc25C was verified at the protein and/or RNA levels. Induction of G1/S cell cycle phase arrest and down-regulation of cell cycle effectors correlated with the up-regulation of miR-221 upon both replicative and oxidative stress-induced senescence. Transient expression of miR-221/222 in HDFs promoted the accumulation of HDFs in G1/S cell cycle phase. We propose that miRNAs up-regulated during replicative senescence may act in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype.

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival.

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.

Beads-on-a-string on a bead: reconstitution and analysis of chromatin on a solid support.

Complex experimental strategies involving in vitro reconstituted chromatin or simple chromatin interaction studies are much facilitated by immobilizing the nucleosomal arrays to paramagnetic beads. Chromatin-containing beads can be retrieved from a reaction mix solution on a magnet fast and quantitatively, effectively separating bound, loosely attached and unbound components efficiently. This chapter details a convenient strategy for immobilization of linear plasmid DNA on streptavidin-coated beads, the reconstitution of chromatin on such beads, and some fundamental handling procedures.

Overexpression of SMARCE1 is associated with CD8+ T-cell infiltration in early stage ovarian cancer.

T-lymphocyte infiltration in ovarian tumors has been linked to a favorable prognosis, hence, exploring the mechanism of T-cell recruitment in the tumor is warranted. We employed a differential expression analysis to identify genes over-expressed in early stage ovarian cancer samples that contained CD8 infiltrating T-lymphocytes. Among other genes, we discovered that TTF1, a regulator of ribosomal RNA gene expression, and SMARCE1, a factor associated with chromatin remodeling were overexpressed in first stage CD8+ ovarian tumors. TTF1 and SMARCE1 mRNA levels showed a strong correlation with the number of intra-tumoral CD8+ cells in ovarian tumors. Interestingly, forced overexpression of SMARCE1 in SKOV3 ovarian cancer cells resulted in secretion of IL8, MIP1b and RANTES chemokines in the supernatant and triggered chemotaxis of CD8+ lymphocytes in a cell culture assay. The potency of SMARCE1-mediated chemotaxis appeared comparable to that caused by the transfection of the CXCL9 gene, coding for a chemokine known to attract T-cells. Our analysis pinpoints TTF1 and SMARCE1 as genes potentially involved in cancer immunology. Since both TTF1 and SMARCE1 are involved in chromatin remodeling, our results imply an epigenetic regulatory mechanism for T-cell recruitment that invites deciphering.

Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer.

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.