In Vitro and Preclinical Systematic Dose-Effect Studies of Auger Electron- and ß Particle-Emitting Radionuclides and External Beam Radiation for Cancer Treatment.

PURPOSE: Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron emitters [99mTc]TcO4- and [123I]I- and ß--particle emitter [188Re]ReO4-. Studies were carried out using genetically modified cells that permitted direct radionuclide comparisons. METHODS AND MATERIALS: Triple-negative MDA-MB-231 breast cancer cells expressing the human sodium iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear absorbed doses using the Medical Internal Radiation Dose (MIRD) formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared with X-ray-treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and sodium iodide symporter-expressing organs was extrapolated to reference human adult models using OLINDA/EXM. RESULTS: [99mTc]TcO4- and [123I]I- reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO4- reduced survival fraction in hNIS-expressing and parental cells. [123I]I- required 2.4- and 1.5-fold lower decays/cell to achieve 37% survival compared with [99mTc]TcO4- and [188Re]ReO4-, respectively, after 72 hours of incubation. Additionally, [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- had superior cell killing effectiveness in vitro compared with X-rays. In vivo, X-ray led to a greater median survival compared with [188Re]ReO4- and [123I]I- (54 days vs 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO4- to a 1 g tumor were 13.8- and 11.2-fold greater than for [123I]I- in female and male models, respectively. CONCLUSIONS: This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- for the first time. We further demonstrate the tumor-controlling effects of [123I]I- and [188Re]ReO4- in comparison with external beam radiation therapy.

Milligram scale enantioresolution of promethazine and its main metabolites, determination of their absolute configuration and assessment of enantioselective effects on human SY-SY5Y cells.

The misuse of pharmaceuticals has significantly increased in recent decades, becoming a major public health concern. The risks associated with medication misuse are particularly high in cases of overdose, especially when the active substances are chiral, as enantioselectivity plays an important role in toxicity. Promethazine (PMZ) is a chiral antihistamine marketed as a racemate and it is misused in "Purple Drank", a recreational drug beverage, that combines codeine and/or PMZ, with soda or alcohol leading to serious health consequences and fatalities in consumers around the world, particularly among teenagers. Information regarding the enantioselectivity in the toxicity of (R,S)-PMZ and its main metabolites, namely promethazine sulfoxide (PMZSO) and desmonomethyl promethazine (DMPMZ), is unknown. This work reported, for the first time, the enantioseparation, in milligram scale, of (R,S)-PMZ, (R,S)-DMPMZ, (R,S)- PMZSO and the determination of their absolute configurations by electronic circular dichroism (ECD). The enantioseparation of all the six enantiomers was accomplished in a homemade semi-preparative column with amylose tris-3,5-dimethylphenylcarbamate (AD) coated with aminopropyl Nucleosil silica. The enantiomeric purity was evaluated using the analytical Lux® 3 µm i-Amylose-3 column, yielding enantiomeric purity values ranging between 94.4% and 99.7%. The elution order of all the enantiomers was accomplished combining the ECD results with an optical rotation detector. The elution order of the enantiomers was influenced only by the chiral selector, rather than the mobile phase. The cytotoxicity of the racemates and the isolated enantiomers towards differentiated SH-SY5Y cells was evaluated. (R,S)-DMPMZ exhibited a significantly higher cytotoxicity than (R,S)-PMZ, suggesting the metabolic bioactivation of (R,S)-PMZ. Conversely, no significant cytotoxicity was found for (R,S)-PMZSO, underscoring a metabolic detoxification pathway. Remarkably, enantioselectivity was observed for the cytotoxicity of PMZ; (R)-PMZ was significantly more cytotoxic than (S)-PMZ. The results underscore the importance to isolate the enantiomers in their enantiomerically form and their correct identification for toxicity enantioselectivity studies, which are vital to understand the drug's behaviour and safety, especially in case of overdoses.

c-di-AMP determines the hierarchical organization of bacterial RCK proteins.

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

Real-World Clinical Characterisation of Polycythaemia Vera Patients from a Prospective Registry in Portugal: Is Resistance to Hydroxyurea a Reality?

Patients with polycythaemia vera (PV) are at increased risk of thrombosis and haemorrhages. Although hydroxyurea (HU) has been the frontline therapy for patients at high risk of vascular complications, about 25% of patients develop resistance/intolerance to this therapy. The aim of this non-interventional, multicentre cohort study was to understand the clinical characteristics and HU treatment response of Portuguese PV patients. HU resistance/intolerance was defined according to adjusted European LeukemiaNet (ELN) criteria. In total, 134 PV patients with a mean (SD) disease duration of 4.8 (5.0) years were included and followed up for 2 years. At baseline, most patients were ≥60 years old (83.2%), at high risk for thrombotic events (87.2%), and receiving HU therapy (79.1%). A total of 10 thrombotic events and 8 haemorrhagic events were reported, resulting in a 5-year probability of thrombo-haemorrhagic events of 17.2%. Haematocrit (p = 0.007), haemoglobin (p = 0.012) and MPN10 symptom score (12.0 (11.6) vs. 10.3 (9.1); p = 0.041) decreased significantly at the 24-month visit compared to baseline. Overall, 75.9% of patients met at least one of the adjusted ELN criteria for HU resistance, and 14.4% of patients remained on HU throughout the study. The results from this real-world study may help identify the subset of patients at higher risk for disease sequelae who may benefit from earlier second-line treatment.

Cardiovascular toxicity in breast cancer patients - contributors and role of cardioprotective drugs.

Breast cancer (BC) patients treated with anthracyclines and/or anti-HER2-targeted therapies (AHT) are highly associated with cardiovascular toxicity (CVT). Our objective was to evaluate the risk of CVT secondary to cancer treatment and the role of cardioprotective-drugs (CPD) in BC patients. We collected a retrospective cohort of females with BC treated with chemotherapy and/or AHT from 2017 to 2019. CVT was defined as LVEF<50% or decline ≥10% during follow-up. As CPD, we considered renin-angiotensin-aldosterone-system inhibitors and beta-blockers. A subgroup analysis of the AHT patients was also performed. A total of 203 women were enrolled. The majority had high or very-high CVT risk score and normal cardiac function at presentation. As for CPD, 35.5% were medicated pre-chemotherapy. All patients were submitted to chemotherapy; AHT were applied to 41.7%. During a 16 months follow-up, 8.5% developed CVT. There was a significant decrease of GLS and LVEF at 12-months (decrease of 1.1% and 2.2%, p<0.001). AHT and combined therapy were significantly associated with CVT. In the AHT sub-group analysis (n=85), 15.7% developed CVT. Patients previously medicated with CPD had a significative lower incidence of CVT (2.9% vs 25.0%, p=0.006). Patients already on CPD presented a higher LVEF at 6-months follow-up (62.5% vs 59.2%, p=0.017). Patients submitted to AHT and anthracycline therapy had higher risk of developing CVT. In the AHT sub-group, pre-treatment with CPD was significantly associated with a lower prevalence of CVT. These results highlight the importance of cardio-oncology evaluation and strengthen the value of primary prevention.

Molecular mechanisms of ferroptosis and their involvement in brain diseases.

Ferroptosis is a type of regulated cell death characterized by intracellular accumulation of iron and reactive oxygen species, inhibition of system Xc-, glutathione depletion, nicotinamide adenine dinucleotide phosphate oxidation and lipid peroxidation. Since its discovery and characterization in 2012, many efforts have been made to reveal the underlying mechanisms, modulating compounds, and its involvement in disease pathways. Ferroptosis inducers include erastin, sorafenib, sulfasalazine and glutamate, which, by inhibiting system Xc-, prevent the import of cysteine into the cells. RSL3, statins, Ml162 and Ml210 induce ferroptosis by inhibiting glutathione peroxidase 4 (GPX4), which is responsible for preventing the formation of lipid peroxides, and FIN56 and withaferin trigger GPX4 degradation. On the other side, ferroptosis inhibitors include ferrostatin-1, liproxstatin-1, α-tocopherol, zileuton, FSP1, CoQ10 and BH4, which interrupt the lipid peroxidation cascade. Additionally, deferoxamine, deferiprone and N-acetylcysteine, by targeting other cellular pathways, have also been classified as ferroptosis inhibitors. Increased evidence has established the involvement of ferroptosis in distinct brain diseases, including Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, multiple sclerosis, and Friedreich's ataxia. Thus, a deep understanding of how ferroptosis contributes to these diseases, and how it can be modulated, can open a new window of opportunities for novel therapeutic strategies and targets. Other studies have shown a sensitivity of cancer cells with mutated RAS to ferroptosis induction and that chemotherapeutic agents and ferroptosis inducers synergize in tumor treatment. Thus, it is tempting to consider that ferroptosis may arise as a target mechanistic pathway for the treatment of brain tumors. Therefore, this work provides an up-to-date review on the molecular and cellular mechanisms of ferroptosis and their involvement in brain diseases. In addition, information on the main ferroptosis inducers and inhibitors and their molecular targets is also provided.

Conserved Double Translation Initiation Site for Δ160p53 Protein Hints at Isoform's Key Role in Mammalian Physiology.

p53 is the most commonly mutated gene in human cancers. Two fundamental reasons for this are its long protein isoforms protect from cancer, while its shorter C-terminal isoforms can support cancer and metastasis. Previously, we have shown that the Δ160p53 protein isoform enhances survival and the invasive character of cancer cells. Here, we identified a translation initiation site nine codons downstream of codon 160-the known initiation codon for the translation of Δ160p53-that is recognized by the translation machinery. When translation failed to initiate from AUG160 due to mutation, it initiated from AUG169 instead, producing similar levels of a similar protein, Δ169p53, which promoted cell survival as efficiently as Δ160p53 following DNA damage. Interestingly, almost all mammalian species with an orthologue to human AUG160 also possess one for AUG169, while none of the non-mammalian species lacking AUG160 have AUG169, even if that region of the p53 gene is well conserved. In view of our findings, we do not believe that Δ169p53 acts as a different p53 protein isoform; instead, we propose that the double translation initiation site strengthens the translation of these products with a critical role in cell homeostasis. Future studies will help verify if this is a more general mechanism for the expression of essential proteins in mammals.

Dabrafenib Plus Trametinib: An Impressive Response in an Adult Patient With BRAF V600E-Mutated and Isocitrate Dehydrogenase (IDH) Wild-Type Glioma.

Key molecular alterations found in the diagnosis and prognosis of brain tumours have been revealed by the latest advances in transcriptomic and genome-wide analysis. In-depth studies revealed that alterations of the V-Raf murine sarcoma viral oncogene homolog B (BRAF) could be shared by different brain tumour types. The identification of BRAF p.V600E mutations in gliomas is nowadays of more importance regarding the development of BRAF-targeted inhibitors. This report presents the case of a 37-year-old female with a voluminous expansive neoplastic lesion, extending from the lenticulocapsular region to the medial aspect of the temporal lobe on the left. Pathological examination revealed an astrocytic neoplasm without high-grade histological features in small biopsy fragments. The molecular study revealed the presence of a mutation in the BRAF V600E gene and CDKN2A/2B homozygous deletion. The lesion was partially removed and irradiated. The patient has been on treatment with dabrafenib plus trametinib for 10 months. In addition to reasonable tolerance, she obtained an impressive tumour reduction, which was manifested in the complete resolution of neurological deficits and in the full acquisition of autonomy. The remarkable results reported in this clinical case justify the pressing need to identify new therapeutic targets in gliomas in the current era of precision medicine.

Metastatic Castration-Resistant Prostate Cancer with BRCA2 Mutation: The Challenge Incorporating PARP Inhibitors and Platinum in Treatment Sequencing.

Prostate cancer is the second most frequent malignancy in men worldwide. Despite the improvement in survival achieved by increasingly early diagnosis and advances in treatment, it is still associated with high mortality. Because of its molecular heterogeneity, there is a need to identify genetic alterations in order to apply targeted therapies. Increasing evidence suggests that the PARP inhibitor olaparib could have a significant synthetic lethal effect in prostate cancer with homologous recombination defects, such as BRCA1/2 mutations. It is not yet known if, under these circumstances, platinum-based chemotherapy induces higher response rates in prostate cancer. We present the case of a patient with BRCA2-mutated metastatic castration-resistant prostate cancer whose treatment sequence included carboplatin and olaparib. LEARNING POINTS: Metastatic castration-resistant prostate cancer (mCRPC) remains a lethal disease despite significant progress in treatment.The BRCA2 mutation is associated with worse survival and so timely genetic screening is important.Studies are needed to identify the best therapeutic sequencing strategy for mCRPC harbouring homologous recombination repair defects, which includes PARP inhibitors and platinum.

Hybrid Silver(I)-Doped Soybean Oil and Potato Starch Biopolymer Films to Combat Bacterial Biofilms.

This study describes the preparation, characterization, and antimicrobial properties of novel hybrid biopolymer materials doped with bioactive silver(I) coordination polymers (bioCPs). Two new bioCPs, [Ag2(µ6-hfa)]n (1) and [Ag2(µ4-nda)(H2O)2]n (2), were assembled from Ag2O and homophthalic (H2hfa) or 2,6-naphthalenedicarboxylic (H2nda) acids as unexplored building blocks. Their structures feature 2D metal-organic and supramolecular networks with 3,6L64 or sql topology. Both compounds act as active antimicrobial agents for producing bioCP-doped biopolymer films based on epoxidized soybean oil acrylate (SBO) or potato starch (PS) as model biopolymer materials with a different rate of degradability and silver release. BioCPs and their hybrid biopolymer films (1@[SBO]n, 2@[SBO]n, 1@[PS]n, and 2@[PS]n) with a very low loading of coordination polymer (0.05-0.5 wt %) show remarkable antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis (Gram-positive) and Escherichia coli and Pseudomonas aeruginosa (Gram-negative) bacteria. Biopolymer films also effectively impair the formation of bacterial biofilms, allowing total biofilm inhibition in several cases. By reporting on new bioCPs and biopolymer films obtained from renewable biofeedstocks (soybean oil and PS), this study blends highly important research directions and widens a limited antimicrobial application of bioCPs and derived functional materials. This research thus opens up the perspectives for designing hybrid biopolymer films with outstanding bioactivity against bacterial biofilms.

Metastatic BRAF V600E-Mutated Thyroid Carcinoma: Molecular/Genetic Profiling Brings a New Therapeutic Option.

We describe a case of a 46-year-old woman diagnosed with localized PTC 20 years ago, having already undergone several treatments with iodine-131 and then treatment with lenvatinib for metastatic disease, to which she developed intolerance. In 2020, in addition to pleural, thoracic, and abdominal lymph node metastasis, progression with symptomatic vertebral bone metastasis was detected, which led to the equation of new therapeutic options. In this context, a genetic/molecular test was carried out, which identified the BRAF V600E mutation and enabled the start of treatment with dabrafenib/trametinib since June 2020. This treatment allowed functional gain, symptomatic relief, and stabilization of the disease. It demonstrates how, in rare tumors, the personalized medicine approach can bring new treatment possibilities.

Relationship of In Vitro Toxicity of Technetium-99m to Subcellular Localisation and Absorbed Dose.

Auger electron-emitters increasingly attract attention as potential radionuclides for molecular radionuclide therapy in oncology. The radionuclide technetium-99m is widely used for imaging; however, its potential as a therapeutic radionuclide has not yet been fully assessed. We used MDA-MB-231 breast cancer cells engineered to express the human sodium iodide symporter-green fluorescent protein fusion reporter (hNIS-GFP; MDA-MB-231.hNIS-GFP) as a model for controlled cellular radionuclide uptake. Uptake, efflux, and subcellular location of the NIS radiotracer [99mTc]TcO4- were characterised to calculate the nuclear-absorbed dose using Medical Internal Radiation Dose formalism. Radiotoxicity was determined using clonogenic and γ-H2AX assays. The daughter radionuclide technetium-99 or external beam irradiation therapy (EBRT) served as controls. [99mTc]TcO4- in vivo biodistribution in MDA-MB-231.hNIS-GFP tumour-bearing mice was determined by imaging and complemented by ex vivo tissue radioactivity analysis. [99mTc]TcO4- resulted in substantial DNA damage and reduction in the survival fraction (SF) following 24 h incubation in hNIS-expressing cells only. We found that 24,430 decays/cell (30 mBq/cell) were required to achieve SF0.37 (95%-confidence interval = [SF0.31; SF0.43]). Different approaches for determining the subcellular localisation of [99mTc]TcO4- led to SF0.37 nuclear-absorbed doses ranging from 0.33 to 11.7 Gy. In comparison, EBRT of MDA-MB-231.hNIS-GFP cells resulted in an SF0.37 of 2.59 Gy. In vivo retention of [99mTc]TcO4- after 24 h remained high at 28.0% ± 4.5% of the administered activity/gram tissue in MDA-MB-231.hNIS-GFP tumours. [99mTc]TcO4- caused DNA damage and reduced clonogenicity in this model, but only when the radioisotope was taken up into the cells. This data guides the safe use of technetium-99m during imaging and potential future therapeutic applications.

Outcomes of Living Kidney Donor Candidates and Living Kidney Recipient Candidates with JC Polyomavirus and BK Polyomavirus Viruria.

INTRODUCTION: Recent data have emerged about a protective association between JCV viruria and chronic kidney disease (CKD). Material and Methods. Single-center retrospective cohort study; 230 living kidney donors (LKD) candidates and 59 potential living kidney receptors (LKR) were enrolled. Plasma and urinary JCV and BKV viral loads were measured in all LKD candidates and in nonanuric LKR candidates. Twenty-six living kidney transplant surgeries were performed. LKR were followed in order to evaluate BKV and JCV viremia and urinary viral shedding after KT. RESULTS: In LKD candidates, JCV viruria was negatively associated with proteinuria of >200 mg/24 hours (JC viruric LKD: 12.5% vs JCV nonviruric LKD: 26.7%, p=0.021, OR:0.393; 95% CI: 0.181-0.854). In a multivariate analysis, LKD candidates with JCV viruria had a lower risk of proteinuria of >200 mg/24 hours (p=0.009, OR: 0.342, 95% CI: 0.153-0.764), in a model adjusted for age, gender, presence of hypertension, and eGFR <80 mL/min. Prevalence of JCV viruria was higher in LKD candidates when compared with LKR candidates (40.0% vs 1.7%, p < 0.001). Among the 26 LKR, 14 (53.8%) KT patients evolved with JCV viruria; 71.4% received a graft from a JCV viruric donor. CONCLUSION: Our data corroborate the recent findings of an eventual protective association between JCV viruria and kidney disease, and we extrapolated this concept to a South European population.

In vitro proof of concept studies of radiotoxicity from Auger electron-emitter thallium-201.

BACKGROUND: Auger electron-emitting radionuclides have potential in targeted treatment of small tumors. Thallium-201 (201Tl), a gamma-emitting radionuclide used in myocardial perfusion scintigraphy, decays by electron capture, releasing around 37 Auger and Coster-Kronig electrons per decay. However, its therapeutic and toxic effects in cancer cells remain largely unexplored. Here, we assess 201Tl in vitro kinetics, radiotoxicity and potential for targeted molecular radionuclide therapy, and aim to test the hypothesis that 201Tl is radiotoxic only when internalized. METHODS: Breast cancer MDA-MB-231 and prostate cancer DU145 cells were incubated with 200-8000 kBq/mL [201Tl]TlCl. Potassium concentration varied between 0 and 25 mM to modulate cellular uptake of 201Tl. Cell uptake and efflux rates of 201Tl were measured by gamma counting. Clonogenic assays were used to assess cell survival after 90 min incubation with 201Tl. Nuclear DNA damage was measured with γH2AX fluorescence imaging. Controls included untreated cells and cells treated with decayed [201Tl]TlCl. RESULTS: 201Tl uptake in both cell lines reached equilibrium within 90 min and washed out exponentially (t1/2 15 min) after the radioactive medium was exchanged for fresh medium. Cellular uptake of 201Tl in DU145 cells ranged between 1.6 (25 mM potassium) and 25.9% (0 mM potassium). Colony formation by both cell lines decreased significantly as 201Tl activity in cells increased, whereas 201Tl excluded from cells by use of high potassium buffer caused no significant toxicity. Non-radioactive TlCl at comparable concentrations caused no toxicity. An estimated average 201Tl intracellular activity of 0.29 Bq/cell (DU145 cells) and 0.18 Bq/cell (MDA-MB-231 cells) during 90 min exposure time caused 90% reduction in clonogenicity. 201Tl at these levels caused on average 3.5-4.6 times more DNA damage per nucleus than control treatments. CONCLUSIONS: 201Tl reduces clonogenic survival and increases nuclear DNA damage only when internalized. These findings justify further development and evaluation of 201Tl therapeutic radiopharmaceuticals.

Long-Term Evolution of SARS-CoV-2 in an Immunocompromised Patient with Non-Hodgkin Lymphoma.

Recent studies have shown that persistent SARS-CoV-2 infections in immunocompromised patients can trigger the accumulation of an unusual high number of mutations with potential relevance at both biological and epidemiological levels. Here, we report a case of an immunocompromised patient (non-Hodgkin lymphoma patient under immunosuppressive therapy) with a persistent SARS-CoV-2 infection (marked by intermittent positivity) over at least 6 months. Viral genome sequencing was performed at days 1, 164, and 171 to evaluate SARS-CoV-2 evolution. Among the 15 single-nucleotide polymorphisms (SNPs) (11 leading to amino acid alterations) and 3 deletions accumulated during this long-term infection, four amino acid changes (V3G, S50L, N87S, and A222V) and two deletions (18-30del and 141-144del) occurred in the virus Spike protein. Although no convalescent plasma therapy was administered, some of the detected mutations have been independently reported in other chronically infected individuals, which supports a scenario of convergent adaptive evolution. This study shows that it is of the utmost relevance to monitor the SARS-CoV-2 evolution in immunocompromised individuals, not only to identify novel potentially adaptive mutations, but also to mitigate the risk of introducing "hyper-evolved" variants in the community. IMPORTANCE Tracking the within-patient evolution of SARS-CoV-2 is key to understanding how this pandemic virus shapes its genome toward immune evasion and survival. In the present study, by monitoring a long-term COVID-19 immunocompromised patient, we observed the concurrent emergence of mutations potentially associated with immune evasion and/or enhanced transmission, mostly targeting the SARS-CoV-2 key host-interacting protein and antigen. These findings show that the frequent oscillation in the immune status in immunocompromised individuals can trigger an accelerated virus evolution, thus consolidating this study model as an accelerated pathway to better understand SARS-CoV-2 adaptive traits and anticipate the emergence of variants of concern.

Methods and techniques for in vitro subcellular localization of radiopharmaceuticals and radionuclides.

In oncology, the holy grail of radiotherapy is specific radiation dose deposition in tumours with minimal healthy tissue toxicity. If used appropriately, injectable, systemic radionuclide therapies could meet these criteria, even for treatment of micrometastases and single circulating tumour cells. The clinical use of α and ß- particle-emitting molecular radionuclide therapies is rising, however clinical translation of Auger electron-emitting radionuclides is hampered by uncertainty around their exact subcellular localisation, which in turn affects the accuracy of dosimetry. This review aims to discuss and compare the advantages and disadvantages of various subcellular localisation methods available to localise radiopharmaceuticals and radionuclides for in vitro investigations.

Bioprospection of Natural Sources of Polyphenols with Therapeutic Potential for Redox-Related Diseases.

Plants are a reservoir of high-value molecules with underexplored biomedical applications. With the aim of identifying novel health-promoting attributes in underexplored natural sources, we scrutinized the diversity of (poly)phenols present within the berries of selected germplasm from cultivated, wild, and underutilized Rubus species. Our strategy combined the application of metabolomics, statistical analysis, and evaluation of (poly)phenols' bioactivity using a yeast-based discovery platform. We identified species as sources of (poly)phenols interfering with pathological processes associated with redox-related diseases, particularly, amyotrophic lateral sclerosis, cancer, and inflammation. In silico prediction of putative bioactives suggested cyanidin-hexoside as an anti-inflammatory molecule which was validated in yeast and mammalian cells. Moreover, cellular assays revealed that the cyanidin moiety was responsible for the anti-inflammatory properties of cyanidin-hexoside. Our findings unveiled novel (poly)phenolic bioactivities and illustrated the power of our integrative approach for the identification of dietary (poly)phenols with potential biomedical applications.

In vitro cytotoxicity of Auger electron-emitting [67Ga]Ga-trastuzumab.

INTRODUCTION: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide. METHODS: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2-negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. RESULTS: Labelling efficiencies for [67Ga]Ga-THP-trastuzumab and [111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/µg (78-92 GBq/µmol). At 4 nM total antibody concentration and 200 × 103 cells/mL, [67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. CONCLUSIONS: [67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.

Inmunotherapy in prostate cancer.

Prostate cancer (PCa) is the second most commonly diagnosed cancer. Although systemic chemotherapeutic agents, such as cabazitaxel, abiraterone and enzalutamde have become available to patients over the last decade, metastatic PCa is still an incurable disease. Immunotherapy is showing great promise in a wide range of other cancer types. To this day, the only immunotherapy approved by the FDA for PCa is the Sipuleucel-T vaccine, which showed significant clinical efficacy. Multiple clinical studies on immunotherapy in PCa are currently underway. OBJECTIVES: Recent clinical trials have shown promising results in immunotherapeutic in treatment for PCa. The authors review previous clinical trials, as well as discuss and emphasize important emerging immunotherapies for PCa. METHODS: Review of the published evidence related to immunotherapy in PCa. PubMed and clinicaltrials.gov databases were used to search for English papers and clinical trials. RESULTS: Multiple clinical trials are testing different immunotherapeuticagents, as well as combinations thereof. The low grade of toxicity associated with these immunotherapies is an appealing advantage for patients, leading to an increased appreciation of theses types of treatments. Until now, only one clinical trial led to a new immunotherapeutic agent to be FDA approved. Importantphase II/III clinical trials are being conducted, and in the near future the concept of PCa treatment might be re-challenged. CONCLUSIONS: Many trials are ongoing to determine the effects of immunotherapy in PCa. These studies may harvest important confirmatory data in the next years, with the potential to reshape PCa treatment.

El cáncer de próstata (CaP) es el segundo cáncer más frecuente. Aunque durante la última década se han hecho disponibles agentes quimioterápicos como Cabazitaxel, Abiraterona y Enzalutamida, el CaP metastásico es todavía una enfermedad incurable. La inmunoterapia está mostrándose muy prometedora en un amplio rango de cánceres de otro tipo. Hasta la fecha, la única inmunoterapia aprobada por la FDA para el CaP es la vacuna Sipuleucel-T, que demostró eficacia clínica significativa. Actualmente, hay múltiples ensayosclínicos de inmunoterapia en CaP en marcha. OBJETIVOS: Ensayos clínicos recientes han mostrado resultadosprometedores de la inmunoterapia en CaP. Los autores revisan los ensayos clínicos previos y también discuten y enfatizan importantes tratamientos inmunoterapéuticos emergentes en CaP.MÉTODOS: Revisión de la evidencia publicada relacionada con inmunoterapia en CaP. Se utilizaron las bases de datos PubMed y clinicaltrials.gov para buscarartículos en inglés y ensayos clínicos. RESULTADOS: Múltiples ensayos clínicos están evaluando diferentes agentes inmunoterapicos, así como combinaciones. El bajo grado de toxicidad asociado con estas inmunoterapias es una ventaja atractiva para los pacientes, que conduce a un aumento de la valoración de este tipo de tratamientos. Hasta ahora, solamente un ensayo clínico ha llevado a la aprobación por la FDAde un nuevo agente inmunoterapico. Se están llevando a cabo importantes ensayos clínicos fase II/III, y en un futuro próximo el concepto de tratamiento del CaP podría ser desafiado de nuevo. CONCLUSIONES: Están en marcha muchos ensayos clínicos para determinar los efectos de la inmunoterapia en CaP. Estos estudios pueden obtener importantesdatos confirmatorios en los próximos años, con el potencial de redefinir el tratamiento del CaP.

Determination of Free-PSA (fPSA) and fPSA/PSA-Ratio Using A Point-of-Care Device.

BACKGROUND: Prostate specific antigen (PSA) and free PSA (fPSA) are important tools for diagnosing prostate cancer (PC). Efforts are continuously undertaken to provide more patient-centered healthcare. The application of point-of-care (POC) systems for laboratory analyses represents a step in this direction. Previous investigations on total PSA measurements using a POC system (concile® Ω100 POC reader) showed good concordance with standard laboratory measurements. For the same POC reader a novel system for fPSA was developed. In the current study, we prospectively evaluated the quality of the POC system for fPSA. METHODS: Sixty-four patients undergoing PSA measurements in our outpatient clinic between 06/2015 and 09/2015 were enrolled in the study. We measured total PSA (tPSA) and fPSA with a POC reader system (concile® Ω100) and a standard laboratory system (Siemens Immulite 2000®) and compared the respective results using linear regression analyses for PSA, fPSA, and fPSA/tPSA ratio (%fPSA). RESULTS: The coefficients of determination (r²) for fPSA and %fPSA were 0.85 (p < 0.001) and 0.82 (p < 0.001) in the subgroup with total PSA between 4 and 10 ng/mL. In the subgroup with tPSA ≤ 4 ng/mL, r² for fPSA concile® was 0.55 (p < 0.001) and 0.10 (p = 0.088) for %fPSA. In the subgroup of tPSA > 10 ng/mL the r² for fPSA and %fPSA was 0.50 (p = 0.022) and 0.50 (p = 0.022), respectively. CONCLUSIONS: The POC fPSA values correlated well with the laboratory analyses, specifically in the clinically relevant diagnostic range of tPSA 4 - 10 ng/mL. These results complement the tPSA data obtained previously and indicate the reliability of the fPSA method and the resulting %fPSA score.