Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

IntroductionThe clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing.Material and methodsIndividual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes.ResultsThe prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients.Conclusion and recommendationOur findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.     

Ability to Generate Patient-Derived Breast Cancer Xenografts Is Enhanced in Chemoresistant Disease and Predicts Poor Patient Outcomes

BackgroundBreast cancer patients who are resistant to neoadjuvant chemotherapy (NeoCT) have a poor prognosis. There is a pressing need to develop in vivo models of chemo resistant tumors to test novel therapeutics. We hypothesized that patient-derived breast cancer xenografts (BCXs) from chemo- naïve and chemotherapy-exposed tumors can provide high fidelity in vivo models for chemoresistant breast cancers.MethodsPatient tumors and BCXs were characterized with short tandem repeat DNA fingerprinting, reverse phase protein arrays, molecular inversion probe arrays, and next generation sequencing.ResultsForty-eight breast cancers (24 post-chemotherapy, 24 chemo-naïve) were implanted and 13 BCXs were established (27%). BCX engraftment was higher in TNBC compared to hormone-receptor positive cancer (53.8% vs. 15.6%, p = 0.02), in tumors from patients who received NeoCT (41.7% vs. 8.3%, p = 0.02), and in patients who had progressive disease on NeoCT (85.7% vs. 29.4%, p = 0.02). Twelve patients developed metastases after surgery; in five, BCXs developed before distant relapse. Patients whose tumors developed BCXs had a lower recurrence-free survival (p = 0.015) and overall survival (p<0.001). Genomic losses and gains could be detected in the BCX, and three models demonstrated a transformation to induce mouse tumors. However, overall, somatic mutation profiles including potential drivers were maintained upon implantation and serial passaging. One BCX model was cultured in vitro and re-implanted, maintaining its genomic profile.ConclusionsBCXs can be established from clinically aggressive breast cancers, especially in TNBC patients with poor response to NeoCT. Future studies will determine the potential of in vivo models for identification of genotype-phenotype correlations and individualization of treatment.     

Single Mutation in Shine-Dalgarno-Like Sequence Present in the Amino Terminal of Lactate Dehydrogenase of Plasmodium Effects the Production of an Eukaryotic Protein Expressed in a Prokaryotic System

One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5′-GGAGGC-3′ sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5′-GGAGGA-3′, by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.     

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.     

Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways

Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzumab-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance.     

Selective genomic copy number imbalances and probability of recurrence in early-stage breast cancer

A number of studies of copy number imbalances (CNIs) in breast tumors support associations between individual CNIs and patient outcomes. However, no pattern or signature of CNIs has emerged for clinical use. We determined copy number (CN) gains and losses using high-density molecular inversion probe (MIP) arrays for 971 stage I/II breast tumors and applied a boosting strategy to fit hazards models for CN and recurrence, treating chromosomal segments in a dose-specific fashion (-1 [loss], 0 [no change] and +1 [gain]). The concordance index (C-Index) was used to compare prognostic accuracy between a training (n = 728) and test (n = 243) set and across models. Twelve novel prognostic CNIs were identified: losses at 1p12, 12q13.13, 13q12.3, 22q11, and Xp21, and gains at 2p11.1, 3q13.12, 10p11.21, 10q23.1, 11p15, 14q13.2-q13.3, and 17q21.33. In addition, seven CNIs previously implicated as prognostic markers were selected: losses at 8p22 and 16p11.2 and gains at 10p13, 11q13.5, 12p13, 20q13, and Xq28. For all breast cancers combined, the final full model including 19 CNIs, clinical covariates, and tumor marker-approximated subtypes (estrogen receptor [ER], progesterone receptor, ERBB2 amplification, and Ki67) significantly outperformed a model containing only clinical covariates and tumor subtypes (C-Index _{full model}, train[test]  =  0.72[0.71] ± 0.02 vs. C-Index _{clinical + subtype model}, train[test]  =  0.62[0.62] ± 0.02; p<10⁻⁶). In addition, the full model containing 19 CNIs significantly improved prognostication separately for ER–, HER2+, luminal B, and triple negative tumors over clinical variables alone. In summary, we show that a set of 19 CNIs discriminates risk of recurrence among early-stage breast tumors, independent of ER status. Further, our data suggest the presence of specific CNIs that promote and, in some cases, limit tumor spread.     

Phenotype sequencing: identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants

Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50-100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a "Phenotype Sequencing" approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110-$340.     

Potential of Raloxifene in reversing osteoarthritis-like alterations in rat chondrocytes: An in vitro model study

The aim of this study was to investigate the effects of Raloxifene (Ral) on degeneration-related changes in osteoarthritis (OA)-like chondrocytes using two- and three-dimensional models. Five-azacytidine (Aza-C) was used to induce OA-like alterations in rat articular chondrocytes and the model was verified at molecular and macrolevels. Chondrocytes were treated with Ral (1, 5 and 10 μM) for 10 days. Caspase-3 activity, gene expressions of aggrecan, collagen II, alkaline phosphatase (ALP), collagen X, matrix metalloproteinases (MMP-13, MMP-3 and MMP-2), and MMP-13, MMP-3 and MMP-2 protein expressions were studied in two-dimensional model. Matrix deposition and mechanical properties of agarose-chondrocyte discs were evaluated in three-dimensional model. One μM Ral reduced expression of OA-related genes, decreased apoptosis, and MMP-13 and MMP-3 protein expressions. It also increased aggrecan and collagen II gene expressions relative to untreated OA-like chondrocytes. In three-dimensional model, 1 μM Ral treatment resulted in increased collagen deposition and improved mechanical properties, although a significant increase for sGAG was not observed. In summation, 1 μM Ral improved matrix-related activities, whereas dose increment reversed these effects except ALP gene expression and sGAG deposition. These results provide evidence that low-dose Ral has the potential to cease or reduce the matrix degeneration in OA.     

RF hydrazine plasma modification of chitosan for antibacterial activity and nanofiber applications

Chitosan nano powders were modified using RF hydrazine plasma produced at low pressure (26.66 Pa) with 13.56 MHz frequency at a power of 100 W for 30 min. Characterization and investigation of the properties of plasma-modified chitosan (PMCh) and non-modified chitosan (Ch) were carried out using an optical monochromator, FTIR, florescence analysis, TGA, SEM, and X-ray techniques. FTIR spectra of PMCh indicated a band broadening at 3436 cm⁻¹ that confirmed increasing functional groups based on H-bonding. The number of NH₂ groups was determined from fluorescence analysis. TGA analysis shows that the moisture absorption is three times higher in the PMCh structure. Ch and PMCh in PVA solutions were used to produce nanofibers by the electrospinning method; average fiber diameters were 480 and 280 nm for Ch and PMCh, respectively. It was found that the antibacterial effect of PMCh is better than the Ch for Gram-positive strains.