Hepatoblastomas with carcinoma features represent a biological spectrum of aggressive neoplasms in children and young adults.

BACKGROUND & AIMS: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the predominant liver cancers in children, though their respective treatment options and associated outcomes differ dramatically. Risk stratification using a combination of clinical, histological, and molecular parameters can improve treatment selection, but it is particularly challenging for tumors with mixed histological features, including those in the recently created hepatocellular neoplasm not otherwise specified (HCN NOS) provisional category. We aimed to perform the first molecular characterization of clinically annotated cases of HCN NOS. METHODS: We tested whether these histological features are associated with genetic alterations, cancer gene dysregulation, and outcomes. Namely, we compared the molecular features of HCN NOS, including copy number alterations, mutations, and gene expression profiles, with those in other pediatric hepatocellular neoplasms, including HBs and HCCs, as well as HBs demonstrating focal atypia or pleomorphism (HB FPAs), and HBs diagnosed in older children (>8). RESULTS: Molecular profiles of HCN NOS and HB FPAs revealed common underlying biological features that were previously observed in HCCs. Consequently, we designated these tumor types collectively as HBs with HCC features (HBCs). These tumors were associated with high mutation rates (∼3 somatic mutations/Mb) and were enriched with mutations and alterations in key cancer genes and pathways. In addition, recurrent large-scale chromosomal gains, including gains of chromosomal arms 2q (80%), 6p (70%), and 20p (70%), were observed. Overall, HBCs were associated with poor clinical outcomes. CONCLUSIONS: Our study indicates that histological features seen in HBCs are associated with combined molecular features of HB and HCC, that HBCs are associated with poor outcomes irrespective of patient age, and that transplanted patients are more likely to have good outcomes than those treated with chemotherapy and surgery alone. These findings highlight the importance of molecular testing and early therapeutic intervention for aggressive childhood hepatocellular neoplasms. LAY SUMMARY: We molecularly characterized a class of histologically aggressive childhood liver cancers and showed that these tumors are clinically aggressive and that their observed histological features are associated with underlying recurrent molecular features. We proposed a diagnostic algorithm to identify these cancers using a combination of histological and molecular features, and our analysis suggested that these cancers may benefit from specialized treatment strategies that may differ from treatment guidelines for other childhood liver cancers.

Physiological and microbiological hormesis in sedge Eleocharis palustris induced by crude oil in phytoremediation of flooded clay soil.

Soil contamination with petroleum hydrocarbons affects plants and rhizospheric microorganisms. Microbial activity participates in important biochemical processes that stimulate, together with plants, the modification of toxic compounds for organisms. A nine-month experiment was set up to study the effect over time of oil on plant height (cm), formation of new plants, plant matter production (gravimetry), and population of rhizospheric microorganisms (serial dilution) in the sedge Eleocharis palustris. Removal of total petroleum hydrocarbons (soxhlet and gravimetry) from the soil was also evaluated. The means of the evaluated variables registered significant statistical differences (Duncan, p < 0.05) regarding the age of the plant and the amount of crude oil. There was a high correlation between oil and plant height (0.848) and with new plants (0.994). 60 mg oil dose promoted the greatest statistical difference in the amounts of roots and plant biomass (p < 0.05). E. palustris exposed to 60 and 75 mg of oil stimulated high densities of microalgae, actinomycetes, fungi, hydrocarbonoclastic bacteria and Pseudomonas spp; the overall ratio was 2:1 relative to natural attenuation. Plant and microorganism variables evaluated registered physiological and microbiological hormetic indices ≥1, showing a positive linear relationship. Natural attenuation was more efficient in removing crude oil. We conclude that E. palustris is tolerant to oil exposure. It is suggested to combine it with natural attenuation for the optimization of soils contaminated with crude oil.

In Silico Analysis of Metabolites from Peruvian Native Plants as Potential Therapeutics against Alzheimer's Disease.

BACKGROUND: Despite research on the molecular bases of Alzheimer's disease (AD), effective therapies against its progression are still needed. Recent studies have shown direct links between AD progression and neurovascular dysfunction, highlighting it as a potential target for new therapeutics development. In this work, we screened and evaluated the inhibitory effect of natural compounds from native Peruvian plants against tau protein, amyloid beta, and angiotensin II type 1 receptor (AT1R) pathologic AD markers. METHODS: We applied in silico analysis, such as virtual screening, molecular docking, molecular dynamics simulation (MD), and MM/GBSA estimation, to identify metabolites from Peruvian plants with inhibitory properties, and compared them to nicotinamide, telmisartan, and grapeseed extract drugs in clinical trials. RESULTS: Our results demonstrated the increased bioactivity of three plants' metabolites against tau protein, amyloid beta, and AT1R. The MD simulations indicated the stability of the AT1R:floribundic acid, amyloid beta:rutin, and tau:brassicasterol systems. A polypharmaceutical potential was observed for rutin due to its high affinity to AT1R, amyloid beta, and tau. The metabolite floribundic acid showed bioactivity against the AT1R and tau, and the metabolite brassicasterol showed bioactivity against the amyloid beta and tau. CONCLUSIONS: This study has identified molecules from native Peruvian plants that have the potential to bind three pathologic markers of AD.

Structural and Functional Analysis of Female Sex Hormones against SARS-CoV-2 Cell Entry.

Emerging evidence suggests that males are more susceptible to severe infection by the SARS-CoV-2 virus than females. A variety of mechanisms may underlie the observed gender-related disparities including differences in sex hormones. However, the precise mechanisms by which female sex hormones may provide protection against SARS-CoV-2 infectivity remains unknown. Here we report new insights into the molecular basis of the interactions between the SARS-CoV-2 spike (S) protein and the human ACE2 receptor. We further report that glycosylation of the ACE2 receptor enhances SARS-CoV-2 infectivity. Importantly, estrogens can disrupt glycan-glycan interactions and glycan-protein interactions between the human ACE2 and the SARS-CoV-2 thereby blocking its entry into cells. In a mouse model of COVID-19, estrogens reduced ACE2 glycosylation and thereby alveolar uptake of the SARS-CoV-2 spike protein. These results shed light on a putative mechanism whereby female sex hormones may provide protection from developing severe infection and could inform the development of future therapies against COVID-19.

A transient increase in MHC-IIlow monocytes after experimental infection with Avibacterium paragallinarum (serovar B-1) in SPF chickens.

Infectious coryza (IC), an upper respiratory tract disease affecting chickens, is caused by Avibacterium paragallinarum. The clinical manifestations of IC include nasal discharge, facial swelling, and lacrimation. This acute disease results in high morbidity and low mortality, while the course of the disease is prolonged and mortality rates are increased in cases with secondary infections. Studies regarding the immune response in infected chickens are scarce, and the local immune response is the focal point of investigation. However, a large body of work has demonstrated that severe infections can impact the systemic immune response. The objective of this study was to evaluate the systemic effects of Avibacterium paragallinarum (serovar B-1) infection on immune cells in specific pathogen-free (SPF) chickens. The current study revealed the presence of a transient circulating monocyte population endowed with high phagocytic ability and clear downregulation of major histocompatibility complex class II (MHC-II) surface expression. In human and mouse studies, this monocyte population (identified as tolerant monocytes) has been correlated with a dysfunctional immune response, increasing the risk of secondary infections and mortality. Consistent with this dysfunctional immune response, we demonstrate that B cells from infected chickens produced fewer antibodies than those from control chickens. Moreover, T cells isolated from the peripheral blood of infected chickens had a lower ability to proliferate in response to concanavalin A than those isolated from control chickens. These findings could be related to the severe clinical signs observed in complicated IC caused by the presence of secondary infections.

An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation.

BACKGROUND: In the poultry industry, quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment. Several assays have been established to evaluate this immunological response in chicken cells. However, these assays have some disadvantages including use of radioactive labels ([3H]-Thymidine assay), necessity of DNA denaturation or digestion (BrdU incorporation assay), lack of sensitivity and underestimation of anti-proliferative effects (MTT assay), and modulation of activation molecules and cell viability reduction (CFSE assay). Overcoming these limitations, the EdU proliferation assay is sensitive and advantageous compared to [3H]-Thymidine radioactive labels in studies on cell proliferation in vitro and allows simultaneous identification of T cell populations. However, this assay has not been established using primary chicken cells to evaluate T cell proliferation by flow cytometry. RESULTS: Here, we established an assay to evaluate the proliferation of primary chicken splenocytes based on the incorporation of a thymidine analog (EdU) and a click reaction with a fluorescent azide, detected by a flow cytometer. We also established a protocol that combines EdU incorporation and immunostaining to detect CD4+ and CD8+ proliferating T cells. By inducing cell proliferation with increasing concentrations of a mitogen (Concanavalin A), we observed a linear increase in EdU positive cells, indicating that our protocol does not present any deficiency in the quantity and quality of reagents that were used to perform the click reaction. CONCLUSIONS: In summary, we established a reliable protocol to evaluate the proliferation of CD4+ and CD8+ chicken T cells by flow cytometry. Moreover, as this is an in-house protocol, the cost per sample using this protocol is low, allowing its implementation in laboratories that process a large number of samples.

Characterization of pediatric hepatocellular carcinoma reveals genomic heterogeneity and diverse signaling pathway activation.

BACKGROUND: Pediatric hepatocellular carcinoma (HCC) is a rare liver tumor in children with a poor prognosis. Comprehensive molecular profiling to understand the underlying genomic drivers of this tumor has not been completed, and it is unclear whether nonfibrolamellar pediatric HCC is more genomically similar to hepatoblastoma or adult HCC. PROCEDURE: To characterize the molecular landscape of these tumors, we analyzed a cohort of 15 pediatric non-FL-HCCs by sequencing a panel of cancer-associated genes and conducting copy-number and gene-expression analyses. RESULTS: We detected multiple types of molecular alterations in Wnt signaling genes, including APC inversion, AMER1 somatic mutation, and most commonly CTNNB1 intragenic deletions. There were multiple alterations to the telomerase pathway via TERT activation or ATRX mutation. Therapeutically targetable activating mutations in MAPK/ERK signaling pathway genes, including MAPK1 and BRAF, were detected in 20% of tumors. TP53 mutations occurred far less frequently in our pediatric HCC cohort than reported in adult cohorts. Tumors arising in children with underlying liver disease were found to be molecularly distinct from the remainder and lacking detectable oncogenic drivers, as compared with those arising in patients without a history of underlying liver disease; the majority of both types were positive for glypican-3, another potential therapeutic target. CONCLUSION: Our study revealed pediatric HCC to be a molecularly heterogeneous group of tumors. Those non-FL-HCC tumors arising in the absence of underlying liver disease harbor genetic alterations affecting multiple cancer pathways, most notably Wnt signaling, and share some characteristics with adult HCC.

Transcultural adaptation and validation of a satisfaction with life scale for Chilean adolescents. / Estudio de adaptación y validación transcultural de una escala de satisfacción con la vida para adolescentes.

BACKGROUND: In recent years, there has been an increasing interest in the determinants of subjecti ve well-being and happiness. In that context, life satisfaction is one of the measures used to assess subjective well-being, with emphasis in adult population. OBJECTIVE: Adapt and validate (cultu rally and linguistically) the Canadian "Satisfaction with Life adapted for Children" (SWLS-C) in Chilean adolescents, in order to assess their global judgement about their own lives. PATIENTS AND METHODS: Cross-sectional study, with adolescents between 10 to 18 years old, of both sexes and three socioeconomic levels in the Metropolitan region of Chile. After a process of translation and back translation, the scale SWLS-C was applied in 3 groups: cognitive interviews (n = 23) to evaluate se mantics, content, reliability and construct validity, a second group was used as pretest (n = 24) and a third for the validation of the test (n = 200). Factor analysis was performed and measurement of convergent and divergent validity. RESULTS: The internal consistency of the scale through Cronbach's alpha was 0.815, with item-total correlations between 0.51 and 0.7. The correlation between scale items varies between 0.390 and 0.607. In the main component analysis, all items met the require ment of a factor greater than 0.3. The inverse correlation between SWLS-C and CDI was significant, with a linear R2 of 0.465. CONCLUSIONS: The Chilean version of the SWLS-C shows adequate psycho metric properties, construct validity, confirming its unidimensional character and the need to keep each item of the Canadian version. SWLS-C, in its Chilean version, is suitable to be self-applied in adolescent population. Further studies are needed to provide further evidence.

Vesiculobullous Cutaneous Larva Migrans in the Absence of Domestic Dogs and Cats. Successful Treatment with Oral Ivermectin.

While conducting research in a protected ecological reserve within Ecuador's subtropical rainforest, a 49-year-old biologist, residing in an Andean city, contracted hookworm-related cutaneous larva migrans (Hr-CLM) in the vesiculobullous clinical form. Since there were no domestic dogs or cats in the reserve, it is likely that wild animals carrying Ancylostoma sp. larvae infected the patient. She was effectively treated with two doses of oral ivermectin, administered 31 days after getting the infection. This case was diagnosed in a temperate city; therefore, a comprehensive travel history and clinical assessments are crucial for an accurate diagnosis and timely treatment.

Metabolic effects of milk fatty acids: A literature review.

Milk and dairy products are known to have a significant role in human development and tissue maintenance due to their high nutritional value. With the higher incidence of obesity and metabolic diseases, nutrition and public health authorities have recommended the intake of fat-free or low-fat dairy due to the saturated fatty acid content of whole-fat products and their effect on serum cholesterol levels. However, recent studies have questioned the association between milk fat consumption and cardiometabolic risk. This literature review aims to compile the scientific evidence of the metabolic effects of milk fatty acids in clinical and basic research studies, as well as their relationship with metabolic disorders and gut microbiota composition. Research shows that various milk fatty acids exert effects on metabolic alterations (obesity, type 2 diabetes and cardiovascular diseases) by modifying glucose homeostasis, inflammation and lipid profile-related factors. Additionally, recent studies have associated the consumption of milk fatty acids with the production of metabolites and the promotion of healthy gut microbiota. From mainly observational studies, evidence suggests that milk and dairy fatty acids are not directly linked to cardiometabolic risk, but further controlled research is necessary to clarify such findings and to assess whether dietary recommendations to choose low-fat dairy foods are necessary for the population for the prevention of obesity and cardiometabolic disease.

T helper type 17 cells contribute to anti-tumour immunity and promote the recruitment of T helper type 1 cells to the tumour.

T helper type 17 (Th17) lymphocytes are found in high frequency in tumour-burdened animals and cancer patients. These lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, have a well-defined role in the development of inflammatory and autoimmune pathologies; however, their function in tumour immunity is less clear. We explored possible opposing anti-tumour and tumour-promoting functions of Th17 cells by evaluating tumour growth and the ability to promote tumour infiltration of myeloid-derived suppressor cells (MDSC), regulatory T cells and CD4(+)  interferon-γ(+) cells in a retinoic acid-like orphan receptor γt (RORγt) -deficient mouse model. A reduced percentage of Th17 cells in the tumour microenvironment in RORγt-deficient mice led to enhanced tumour growth, that could be reverted by adoptive transfer of Th17 cells. Differences in tumour growth were not associated with changes in the accumulation or suppressive function of MDSC and regulatory T cells but were related to a decrease in the proportion of CD4(+) T cells in the tumour. Our results suggest that Th17 cells do not affect the recruitment of immunosuppressive populations but favour the recruitment of effector Th1 cells to the tumour, thereby promoting anti-tumour responses.

Bartonella spp. in households with cats: Risk factors for infection in cats and human exposure.

The aim of this study was to estimate the occurrence of Bartonella spp. per household in cats and the risk factors for Bartonella spp. positivity in cats and their owners from Valdivia, Chile. A total of 464 cats (distributed within 324 households) and 326 humans (control group [n = 112] and cat owner [n = 214]) distributed in 262 households were sampled. From the cat owners (n = 214), 128 humans were in households where the cat was also sampled, totaling 84 households with dual sampling. Real-time PCR (qPCR) was used for Bartonella spp. detection in blood from cats and humans, and immunofluorescent immunoassay (IFA) anti-Bartonella henselae was performed in human serum samples. Out of the total of 324 households, 20.43% presented at least one Bartonella positive cat. From the households with dual sampling, 29.7% (25/84) presented at least one qPCR-Bartonella spp. positive cat. However, Bartonella DNA was not amplified in humans, and in 7.3% (6/82) of the households was found at least one of the cat's owners exposed to B. henselae. Cats younger than one year (Odds Ratio (OR) = 5.3), non-neutered (OR 3.46), sampled at home (OR 5.82), and with improper application of tick/flea control products (OR 3.13) showed a higher risk for Bartonella spp. presence. Humans with occupational exposure involving animal contact, were more likely to exhibit B. henselae seropositivity (OR 7.5). Bartonella spp. was present in the cats a moderate number of households, but Bartonella DNA was not detected in owners' blood, inferring that there is a low risk of recent human infection in the studied population.

Co-occurring pathogenic variants in 6q27 associated with dementia spectrum disorders in a Peruvian family.

Evidence suggests that there may be racial differences in risk factors associated with the development of Alzheimer's disease and related dementia (ADRD). We used whole-genome sequencing analysis and identified a novel combination of three pathogenic variants in the heterozygous state (UNC93A: rs7739897 and WDR27: rs61740334; rs3800544) in a Peruvian family with a strong clinical history of ADRD. Notably, the combination of these variants was present in two generations of affected individuals but absent in healthy members of the family. In silico and in vitro studies have provided insights into the pathogenicity of these variants. These studies predict that the loss of function of the mutant UNC93A and WDR27 proteins induced dramatic changes in the global transcriptomic signature of brain cells, including neurons, astrocytes, and especially pericytes and vascular smooth muscle cells, indicating that the combination of these three variants may affect the neurovascular unit. In addition, known key molecular pathways associated with dementia spectrum disorders were enriched in brain cells with low levels of UNC93A and WDR27. Our findings have thus identified a genetic risk factor for familial dementia in a Peruvian family with an Amerindian ancestral background.

Protocol to assess the effects of dysfunctional human vascular smooth muscle cells on other brain cells using in vitro models of Alzheimer's disease.

Here, we present a protocol to culture primary human vascular smooth muscle cells (VSMCs) under Alzheimer's disease (AD)-like conditions, including steps for morphological characterization with microscopy. We then describe functional assays, including wound healing, transwell, coculture, and supernatant assays, to evaluate the effect of dysfunctional VSMCs on the induction of the AD-associated microglial phenotype. Our approach can be applied to assess the effects of dysfunctional VSMCs on other cerebral cell lines including pericytes, astrocytes, and neurons under AD-like conditions in vitro. For complete details on the use and execution of this protocol, please refer to Aguilar-Pineda et al. (2021).

A Validation Framework for Somatic Copy Number Detection in Targeted Sequencing Panels.

Somatic copy number alterations (SCNAs) in tumors are clinically significant diagnostic, prognostic, and predictive biomarkers. SCNA detection from targeted next-generation sequencing panels is increasingly common in clinical practice; however, detailed descriptions of optimization and validation of SCNA pipelines for small targeted panels are limited. This study describes the validation and implementation of a tumor-only SCNA pipeline using CNVkit, augmented with custom modules and optimized for clinical implementation by testing reference materials and clinical tumor samples with different classes of copy number variation (CNV; amplification, single copy loss, and biallelic loss). Using wet-bench and in silico methods, various parameters impacting CNV calling, including assay-intrinsic variables (establishment of normal reference and sequencing coverage), sample-intrinsic variables (tumor purity and sample quality), and CNV algorithm-intrinsic variables (bin size), were optimized. The pipeline was trained and tested on an optimization cohort and validated using an independent cohort with a sensitivity and specificity of 100% and 93%, respectively. Using custom modules, intragenic CNVs with breakpoints within tumor suppressor genes were uncovered. Using the validated pipeline, re-analysis of 28 pediatric solid tumors that had been previously profiled for mutations identified SCNAs in 86% (24/28) samples, with 46% (13/28) samples harboring findings of potential clinical relevance. Our report highlights the importance of rigorous establishment of performance characteristics of SCNA pipelines and presents a detailed validation framework for optimal SCNA detection in targeted sequencing panels.

A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Chromosome 14q loss defines a molecular subtype of clear-cell renal cell carcinoma associated with poor prognosis.

Loss of chromosome 14 has been associated with poor outcomes in clear-cell renal cell carcinoma. Expression of HIFα isoforms has been linked to distinct molecular phenotypes of clear-cell renal cell carcinoma. We hypothesized that chromosome 14 loss could lead to a decrease in HIF1α levels, as its gene (HIF1A) resides in this chromosome. We analyzed 112 archival clear-cell renal cell carcinoma tumor specimens with 250K SNP microarrays. We also evaluated expression of HIFα isoforms by qPCR and immunohistochemistry in a subset of 30 patients. Loss of chromosome 14q was associated with high stage (III-IV, P=0.001), high risk for recurrence (P=0.002, RR 2.78 (1.506-5.153)) and with decreased overall survival (P=0.030) in non-metastatic clear-cell renal cell carcinoma. HIF1α mRNA and protein expression was reduced in specimens with loss of 14q (P=0.014) whereas HIF2α was not. Gain of 8q was associated with decreased overall survival (P<0.0001). Our studies confirm an association between 14q loss and clinical outcome in non-metastatic clear-cell renal cell carcinoma patients and that 8q gain is a candidate prognostic marker for decreased overall survival and appears to further decrease survival in patients with 14q loss. We have also identified that differential expression of HIF1α is associated with 14q loss. Further exploration of 8q gain, 14q loss, MYC, HIF1A and EPAS1 (HIF2α) as molecular markers of tumor behavior and prognosis could aid in personalizing medicine for patients with clear-cell renal cell carcinoma.

[Cystic neoplasm of the pancreas: a ten years experience]. / Tumores quísticos de páncreas. Informe de 10 años.

BACKGROUND: Pancreatic cystic lesions (PCL) are identified in 1 % of patients who undergo abdominal computed tomography scans, because it is difficult to discriminate clinically between benign and malignant PCL. The PCL must be distinguished from inflammatory pseudocysts, which can have a similar radiographic appearance. The aim of this study was to review the incidence of PCL during 10 years. METHODS: Nineteen consecutive patients who underwent surgical resection of a cystic lesion of the pancreas during a 10-year period were reviewed. RESULTS: From 1998, 434 patients underwent to pancreatic surgery, 103 (25.75%) resulted with neoplasm of the pancreas and a PCL was diagnosed in 19 of them (18.44%). They were more common in women (n=15, 79%). The pathologic diagnosis was mucinous cystadenoma (n=6, 31.5%) followed by serous cystadenoma and solid cystic papilar tumor (n=4, 21%). Mucinous cystadenocarcinoma was diagnosed in a 77 year-old man and a serous cystadenocarcinoma was diagnosed in a 53 years-old woman; cystic degeneration of two insulinomas were diagnosed in a 71 year-old woman and a 32 year-old man (5.26%). CONCLUSIONS: PCLs represent a spectrum of associated diseases. The incidence of serous cystadenoma is lower in our experience.

Antibodies induced by oral immunization of mice with a recombinant protein produced in tobacco plants harboring Bordetella pertussis epitopes.

Bordetella pertusis causes whooping cough or pertussis, disease that has not been eradicated and is reemerging despite the availability and massive application for decades of vaccines, such as Boostrix® which is an acellular vaccine harboring two regions of S1 subunit of the pertussis toxin, one region of filamentous hemagglutinin and one region of pertactin. In 2008, the World Health Organization estimated 16 million new cases and 95% occurred in developing countries with 195,000 children's deaths. We attempt to improve the vaccine against whooping cough and reduce its production costs by obtaining plants and bacteria expressing a heterologous protein harboring pertactin, pertussis toxin, and filamentous hemagglutinin epitopes from B. pertussis and assessing its immunogenicity after oral administration to mice. First, we designed a synthetic gene that encodes a multiepitope, then it was cloned into a vector for transient transformation by infiltration of tobacco plants with low amounts of nicotine; the codon bias-optimized construct was also cloned into an Escherichia coli expression vector. Recombinant proteins from E. coli cells (PTF) and tobacco leaves (PTF-M3') were purified by nickel affinity with a yield of 0.740 mg of recombinant protein per g dry weight. Purified recombinant proteins were administered orally to groups of Balb/c mice using the Boostrix® vaccine and vehicle (PBS) as positive and negative controls, respectively. A higher mucosal and systemic antibody responses were obtained in mice receiving the PTF and PTF-M3' proteins than Boostrix® or PBS. These findings prove the concept that oral administration of multiepitope recombinant proteins expressed in plants may be a potential edible vaccine. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02107-1.

PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.

PTEN haploinsufficiency is common in hormone-sensitive prostate cancer, though the incidence of genomic deletion and its downstream effects have not been elucidated in clinical samples of hormone refractory prostate cancer (HRPC). Progression to androgen independence is pivotal in prostate cancer and mediated largely by the androgen receptor (AR). Since this process is distinct from metastatic progression, we examined alterations of the PTEN gene in locally advanced recurrent, non-metastatic human HRPC tissues. Retrospective analyses of PTEN deletion status were correlated with activated downstream phospho-Akt (p-Akt) pathway proteins and with the androgen receptor. The prevalence of PTEN genomic deletions in transurethral resection samples of 59 HRPC patients with known clinical outcome was assessed by four-colour FISH analyses. FISH was performed using six BAC clones spanning both flanking PTEN genomic regions and the PTEN gene locus, and a chromosome 10 centromeric probe. PTEN copy number was also evaluated in a subset of cases using single nucleotide polymorphism (SNP) arrays. In addition, the samples were immunostained with antibodies against p-Akt, p-mTOR, p-70S6, and AR. The PTEN gene was deleted in 77% of cases, with 25% showing homozygous deletions, 18% homozygous and hemizygous deletions, and 34% hemizygous deletions only. In a subset of the study group, SNP array analysis confirmed the FISH findings. PTEN genomic deletion was significantly correlated to the expression of downstream p-Akt (p < 0.0001), AR (p = 0.025), and to cancer-specific mortality (p = 0.039). PTEN deletion is common in HRPC, with bi-allelic loss correlating to disease-specific mortality and associated with Akt and AR deregulation.