Biophysical studies do not reveal direct interactions between human PF4 and Ad26.COV2.S vaccine.

BACKGROUND: COVID-19 vaccines have been widely used to control the SARS-CoV-2 pandemic. In individuals receiving replication-incompetent, adenovirus vector-based COVID-19 vaccines (eg, ChAdOx1 nCoV-19 [AstraZeneca] or Ad26.COV2.S [Johnson & Johnson/Janssen] vaccines), a very rare but serious adverse reaction has been reported and described as vaccine-induced immune thrombotic thrombocytopenia (VITT). The exact mechanism of VITT following Ad26.COV2.S vaccination is under investigation. Antibodies directed against human platelet factor 4 (PF4) are considered critical in the pathogenesis of VITT, suggesting similarities with heparin-induced thrombocytopenia. It has been postulated that components of these vaccines mimic the role of heparin by binding to PF4, triggering production of these anti-PF4 antibodies. OBJECTIVES: This study aimed to investigate the potential interaction between human PF4 and Ad26.COV2.S vaccine using several biophysical techniques. METHODS: Direct interaction of PF4 with Ad26.COV2.S vaccine was investigated using dynamic light scattering, biolayer interferometry, and surface plasmon resonance. For both biosensing methods, the Ad26.COV2.S vaccine was immobilized to the sensor surface and PF4 was used as analyte. RESULTS: No direct interactions between PF4 and Ad26.COV2.S vaccine could be detected using dynamic light scattering and biolayer interferometry. Surface plasmon resonance technology was shown to be unsuitable to investigate these types of interactions. CONCLUSION: Our findings make it very unlikely that direct binding of PF4 to Ad26.COV2.S vaccine or components thereof is driving the onset of VITT, although the occurrence of such interactions after immunization (potentially facilitated by unknown plasma or cellular factors) cannot be excluded. Further research is warranted to improve the understanding of the full mechanism of this adverse reaction.

Effect of Laminaria digitata dietary inclusion and CAZyme supplementation on blood cells, serum metabolites and hepatic lipids and minerals of weaned piglets.

Seaweeds, such as Laminaria digitata, are a sustainable alternative to conventional feedstuffs for weaned piglet diets, improving their health and mitigating environmental impacts. L. digitata has a complex cell wall that can be difficult for monogastrics to digest. However, carbohydrate-active enzymes (CAZymes) such as Rovabio® Excel AP and alginate lyase can help break down these polysaccharides and render intracellular nutrients more accessible. This study aimed to evaluate the impact of 10% L. digitata feed inclusion and CAZyme supplementation on piglet blood cells, serum metabolites, liver lipid and mineral profiles. Forty weaned piglets were randomly assigned to one of four diets (n = 10 each): a control diet, 10% L. digitata (LA), 10% L. digitata + 0.005% Rovabio® Excel AP (LAR), and 10% L. digitata + 0.01% alginate lyase (LAL). After two weeks of trial, animals were slaughtered and liver and blood serum samples taken for analysis. The results showed that the LA and LAL diets increased blood lymphocytes, IgG and IgM, and decreased serum lipids, improving both cellular and humoral immune response and cardiovascular health. Dietary CAZymes reversed the anti-inflammatory and hematopoietic effects. Additionally, cortisol levels were reduced with seaweed inclusion compared to the control diet (P < 0.001). In the liver, total n-3 PUFA and n-6/n-3 ratio were increased and decreased, respectively, due to eicosapentaenoic acid and α-linolenic acid accumulation (P < 0.001). However, total liver mineral content was incorporated to a lesser extent with the combined seaweed and enzyme diets (P < 0.001), potentially indicating a negative effect on mineral bioavailability. Overall, results suggest that a 10% L. digitata inclusion can effectively improve piglet health by reducing stress during weaning, without the need for dietary CAZymes.

Influence of Dietary Supplementation with an Amino Acid Mixture on Inflammatory Markers, Immune Status and Serum Proteome in LPS-Challenged Weaned Piglets.

In order to investigate the effect of a dietary amino acid mixture supplementation in lipopolysaccharide (LPS)-challenged weaned piglets, twenty-seven 28-day-old (8.2 ± 1.0 kg) newly weaned piglets were randomly allocated to one of three experimental treatments for five weeks. Diet 1: a CTRL treatment. Diet 2: an LPS treatment, where piglets were intraperitoneally administered LPS (25 µg/kg) on day 7. Diet 3: an LPS+MIX treatment, where piglets were intraperitoneally administered LPS on day 7 and fed a diet supplemented with a mixture of 0.3% of arginine, branched-chain amino acids (leucine, valine, and isoleucine), and cystine (MIX). Blood samples were drawn on day 10 and day 35, and serum was analysed for selected chemical parameters and proteomics. The LPS and LPS+MIX groups exhibited an increase in haptoglobin concentrations on day 10. The LPS group showed an increased cortisol concentration, while this concentration was reduced in the LPS+MIX group compared to the control group. Similarly, the LPS+MIX group showed a decreased haptoglobin concentration on day 35 compared to the two other groups. Immunoglobulin concentrations were affected by treatments. Indeed, on day 10, the concentrations of IgG and IgM were decreased by the LPS challenge, as illustrated by the lower concentrations of these two immunoglobulins in the LPS group compared to the control group. In addition, the supplementation with the amino acid mixture in the LPS+MIX further decreased IgG and increased IgM concentrations compared to the LPS group. Although a proteomics approach did not reveal important alterations in the protein profile in response to treatments, LPS-challenged piglets had an increase in proteins linked to the immune response, when compared to piglets supplemented with the amino acid mixture. Overall, data indicate that LPS-challenged piglets supplemented with this amino acid mixture are more protected against the detrimental effects of LPS.

Glutamine and cystine-enriched diets modulate aquaporins gene expression in the small intestine of piglets.

The regulation of glycerol permeability in the gastrointestinal tract is crucial to control fat deposition, lipolysis and gluconeogenesis. Knowing that the amino acid glutamine is a physiological regulator of gluconeogenesis, whereas cystine promotes adiposity, herein we investigated the effects of dietary supplementation with glutamine and cystine on the serum biochemical parameters of piglets fed on amino acid-enriched diets, as well as on the transcriptional profile of membrane water and glycerol channels aquaporins (AQPs) in the ileum portion of the small intestine and its impact on intestinal permeability. Twenty male piglets with an initial body weight of 8.8 ± 0.89 kg were allocated to four dietary treatments (n = 5) and received, during a four week-period, a basal diet without supplementation (control) or supplemented with 8 kg/ton of glutamine (Gln), cystine (Cys) or the combination of the two amino acids in equal proportions (Gln + Cys). Most biochemical parameters were found improved in piglets fed Gln and Cys diet. mRNA levels of AQP3 were found predominant over the others. Both amino acids, individually or combined, were responsible for a consistent downregulation of AQP1, AQP7 and AQP10, without impacting on water permeability. Conversely, Cys enriched diet upregulated AQP3 enhancing basolateral membranes glycerol permeability and downregulating glycerol kinase (GK) of intestinal cells. Altogether, our data reveal that amino acids dietary supplementation can modulate intestinal AQPs expression and unveil AQP3 as a promising target for adipogenesis regulation.

Factors associated with the aggressiveness of care at the end of life for patients with cancer dying in hospital: a nationwide retrospective cohort study in mainland Portugal.

INTRODUCTION: There is growing concern about the aggressiveness of cancer care at the end of life (ACCEoL), defined as overly aggressive treatments that compromise the quality of life at its end. Recognising the most affected patients is a cornerstone to improve oncology care. Our aim is to identify factors associated with ACCEoL for patients with cancer dying in hospitals. METHODS: All adult patients with cancer who died in public hospitals in mainland Portugal (January 2010 to December 2015), identified from the hospital morbidity database. This database provided individual clinical and demographic data. We obtained hospital and region-level variables from a survey and National Statistics. The primary outcome is a composite ACCEoL measure of 16 indicators. We used multilevel random effects logistic regression modelling (p<0·05). RESULTS: We included 92 155 patients: median age 73 years; 62% male; 53% with metastatic disease. ACCEoL prevalence was 71% (95% CI 70% to 71%). The most prevalent indicators were >14 days in the hospital (43%, 42-43) and surgery (28%, 28-28) in the last 30 days. Older age (p<0·001), breast cancer (OR 0·83; 95% CI 0·76 to 0·91), and metastatic disease (0·54; 95% CI 0·50 to 0·58) were negatively associated with ACCEoL. In contrast, higher Deyo-Charlson Comorbidity Index (p<0·001), gastrointestinal and haematological malignancies (p<0·001), and death at cancer centre (1·31; 95% CI 1·01 to 1·72) or hospital with medical oncology department (1·29; 95% CI 1·02 to 1·63) were positively associated with ACCEoL. There was no association between hospital palliative care services at the hospital of death and ACCEoL. CONCLUSION: Clinical factors related to a better understanding of disease course are associated with ACCEoL reduction. Patients with more comorbidities and gastrointestinal malignancies might represent groups with complex needs, and haematological patients may be at increased risk because of unpredictable prognosis. Improvement of hospital palliative care services could help reduce ACCEoL, particularly in cancer centres and hospitals with medical oncology department, as those services are usually under-resourced, thus reaching few.

A Potential Link Between Prolonged Cork Exposure and Intestinal-Type Sinonasal Adenocarcinoma - Special Findings of a Retrospective Cohort Analysis.

INTRODUCTION: intestinal-type sinonasal adenocarcinoma (ITAC) is a rare epithelium tumor of the nasal cavities and paranasal sinuses. Exposure to wood and leather dusts is a strong etiological factor related to its development. Prolonged cork exposure has rarely been associated. MATERIALS AND METHODS: thirty-seven-year (1981-2018) retrospective cohort analysis of all consecutive patients with sinonasal cancer (SNC) followed at our institution. Medical records were reviewed to determine patient demographics, occupational/environmental exposure, location and extent of the tumor, stage, histopathology findings, treatment strategies, and oncologic outcomes. Survival analysis was done using Kaplan-Meier method. RESULTS: we evaluated 379 patients with SNC, including 39 (10.3%) ITAC. Patient median age was 73 years (range 49-87), 56% male and 69% with identified professional occupational exposure (54% for cork; 69.2% considering only those for which an agent has been identified). Seventy-two percent had locally advanced disease (stage III or IVA-B). The initial treatment was surgery in 77%, and 54% received adjuvant radiotherapy. The median time to progression, progression-free survival, and overall-survival was 2.36 years (95% CI 1.54-8.70), 1.96 years (95% CI 1.43-3.74), and 3.51 years (95% CI 2.33-10.02), respectively. CONCLUSION: ITAC is an uncommon malignancy that grows silently, which contributes to delayed diagnosis, advanced stage and low survival rates. In our cohort, we observed a high prevalence of cork occupational exposure. This finding may lead to the implementation of protection measures and suggest a potential link to be further studied.

Tumor Cell Attack by Crotalicidin (Ctn) and Its Fragment Ctn[15-34]: Insights into Their Dual Membranolytic and Intracellular Targeting Mechanism.

Crotalicidin (Ctn) and its fragment Ctn[15-34] are snake-venom-derived, cathelicidin-related peptides outstanding for their promising antimicrobial, antifungal, and antitumoral properties. In this study, we describe their membranolytic mechanisms as well as their putative interference with intracellular targets, both contributing to their antitumoral action against a pro-monocytic leukemia cell line. Initial flow cytometry assays demonstrated peptide ability to induce tumor cell membrane permeabilization and caspase-dependent apoptosis, without total activity reduction by serum proteases up to 24 h (Ctn) and 18 h (Ctn[15-34]). In addition, both Ctn and Ctn[15-34] showed preference for tumor cells rather than healthy cells, with selectivity ratios (tumoral vs healthy cells) of 17 and 7, respectively. Further microscopy and flow cytometry studies suggested their preferential accumulation in the cytoplasmic membrane and nucleus and proposed multiple predominant routes of peptide uptake, including direct entry and endocytosis. Affinity purification followed by proteomic identification experiments revealed both peptides to interact with proteins involved in DNA and protein metabolism, cell cycles, signal transduction, and/or programmed cell death, among others. These results suggest a putative role of Ctn and Ctn[15-34] to interact with key intracellular pathways, ultimately contributing to tumor cell death by necrosis/apoptosis. Altogether, this work proposes a dual mechanism underlying the antitumoral activity of Ctn and Ctn[15-34] and reinforces their potential as future therapeutic drugs.

Low rectal cancer treatment strategies: a cohort study assessing watch and wait.

PURPOSE: Treatment strategies for low rectal cancer have been evolving toward achieving less treatment morbidity with the same oncological success-we aimed to assess the results of the new watch and wait (W&W) strategy in our cohort. METHODS: A tertiary care cohort study was conducted. New patients with rectal adenocarcinoma up to 6 cm from the anal margin, cM0, locally staged higher than cT1N0, evaluated between November 2014 and October 2018, were included. All 93 patients received neoadjuvant radiotherapy ± chemotherapy. Re-evaluation was planned 8-12 weeks after the end of treatment. Patients showing clinical complete response (cCR) were given the choice of either to proceed to surgery or to enter W&W. RESULTS: Of the 93 patients, 82.8% were re-evaluated and 20.8% had cCR. Patients in clinical stages II/III were significantly less likely to achieve cCR than those in stage I (p = 0.017). After a mean follow-up of 17.44 months, there were 4 regrowths in the 16 patients under W&W, all submitted to R0 surgery, ypN0; there were no deaths or local recurrences; one patient with regrowth had distant recurrence. Sixty patients underwent direct surgery after a mean follow-up of 16.23 months; 3 patients had local and distant recurrences; 7 others had only distant recurrences; there were 8 deaths. There were no statistically significant differences between patients under W&W and patients who underwent direct surgery regarding local or distant recurrences, or death (p > 0.9; p = 0.44; p = 0.19, respectively). CONCLUSION: The W&W strategy for low rectal cancer achieved the same oncological outcomes as the traditional strategy while sparing some patients from surgery.

ATP1A3: an unusual antigen to consider in patients with subacute ataxia and vertical gaze palsy.

The anti-ATP1A3 antibody seems to be directed against a neuronal and cardiac surface protein of the proton ATPase. It was recently identified in a patient with a paraneoplastic syndrome characterized by cerebellar, neuro-ophthalmologic and other neurological symptoms. We report another patient with a similar clinical and radiological syndrome but with a different associated tumor and outcome.

EMA Review of Daunorubicin and Cytarabine Encapsulated in Liposomes (Vyxeos, CPX-351) for the Treatment of Adults with Newly Diagnosed, Therapy-Related Acute Myeloid Leukemia or Acute Myeloid Leukemia with Myelodysplasia-Related Changes.

On June 28, 2018, the Committee for Medicinal Products for Human Use adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Vyxeos, intended for the treatment of acute myeloid leukemia (AML). Vyxeos was designated as an orphan medicinal product on January 11, 2012. The applicant for this medicinal product was Jazz Pharmaceuticals Ireland Limited. Vyxeos is a liposomal formulation of a fixed combination of daunorubicin and cytarabine, antineoplastic agents that inhibit topoisomerase II activity and also cause DNA damage. The strength of Vyxeos is 5 units/mL, where 1 unit equals 1.0 mg cytarabine plus 0.44 mg daunorubicin. The marketing authorization holder Jazz Pharmaceuticals had found that this was an optimal ratio for the efficacy of the product. Study CLTR0310-301, a phase III, multicenter, randomized, trial of Vyxeos (daunorubicin-cytarabine) liposome injection versus standard 3+7 daunorubicin and cytarabine in patients aged 60-75 years with untreated high-risk (secondary) AML, showed a statistically significant difference between the two groups in overall survival (OS) with a median OS of 9.56 months in the daunorubicin-cytarabine arm compared with 5.95 months for standard chemotherapy (hazard ratio, 0.69; 95% confidence interval, 0.52-0.90; one-sided p = .003). The most common side effects were hypersensitivity including rash, febrile neutropenia, edema, diarrhea/colitis, mucositis, fatigue, musculoskeletal pain, abdominal pain, decreased appetite, cough, headache, chills, arrhythmia, pyrexia, sleep disorders, and hypotension. IMPLICATIONS FOR PRACTICE: Vyxeos has demonstrated a clinically significant improvement in overall survival compared with the standard of care 7+3 in the proposed population of patients with newly diagnosed acute myeloid leukemia (AML) with myelodysplasia-related changes and therapy-related AML. This is remarkable given the very poor prognosis of these patients and their unmet medical need. Secondary endpoints support the primary outcome, in particular an increased rate of hematopoietic stem cell transplantation, which is potentially the only curative treatment in AML.

Colon Adenocarcinoma Stage IIA-Can We Predict Relapse?

PURPOSE: To determine prognostic factors for stage IIA colon cancer (CC) recurrence in patients undergoing curative intent surgery without adjuvant treatment. METHODS: Single-centre cohort study. All patients with stage IIA CC discussed in a multidisciplinary colorectal cancer clinic from January 2010 to December 2012 were evaluated. Clinical data, laboratory data and tumour features, including expression of DNA repair proteins (EDRP), were analysed. Assessment of overall and disease free survival, recurrence, recurrence site and recurrence's method of diagnosis was performed. The associations between variables were tested through the Fisher's exact test (SPSS 23). RESULTS: Fifty-five patients were included (55% male gender; mean age at diagnosis was 70.3 years (42-88)). CC was in the left colon in 62%, high grade in 7% and had lymphovascular invasion in 7% of the cases. Only one patient was submitted to emergent surgery for obstructive symptoms. In 55% of cases ≥ 12 lymph nodes were collected. There was EDRP loss in nine patients (MLH1/PMS2: six; MSH2/MSH6: three)-only two fulfilled revised Bethesda criteria. Recurrence occurred in five patients (8.9%), and it was diagnosed through surveillance in all of them. No variable showed a statistically significant association with recurrence; however, there were no recurrences in patients with EPRD loss (p = 0.209). Mean follow-up time was 43 months (2-70). In those with recurrence, mean disease-free survival was 23.4 months. CONCLUSIONS: The overall good prognosis and absence of recurrence predictive factors were confirmed, validating the decision of not to submit stage IIA CC patients to chemotherapy risks.

Lapatinib with ECF/X in the first-line treatment of metastatic gastric cancer according to HER2neu and EGFR status: a randomized placebo-controlled phase II study (EORTC 40071).

PURPOSE: HER2-targeted therapy with trastuzumab and (CF/X) prolonged overall survival (OS) in metastatic HER2neu+ gastric carcinoma (GC). Lapatinib inhibits both EGFR and HER2neu. We investigated the efficacy and safety of lapatinib with epirubicin (E) + CF/X in GC according to HER2neu and EGFR status. METHODS: Tumors from chemotherapy-naïve patients were screened centrally by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Patients with EGFR and/or HER2neu expression or amplification were allocated to three strata based on EGFR/HER2neu status and were randomized to lapatinib (arm A) or placebo (arm B), with 6 cycles of ECF or ECX (investigator-selected). The primary endpoint was progression-free survival (PFS) in stratum 3. RESULTS: 29 of 72 screened patients were randomized to strata 1 (HER2neu+: by FISH and IHC, n = 6), 2 (HER2neu-: by FISH/+ by IHC, n = 5) and 3 (HER2neu-/EGFR+, n = 18), of which 28 patients were eligible (14 per arm). Enrollment was curtailed after announcement of the negative LOGiC trial results. Median PFS was 8.0 versus 5.9 months (HR = 0.86, 95% CI 0.37-1.99) in the per protocol population, and 8.0 versus 6.3 months (HR = 0.85, 95% CI 0.30-2.46) for stratum 3, in the lapatinib versus placebo arm respectively. Median OS was 13.8 versus 10.1 months, respectively (HR = 0.90, 95% CI 0.35-2.27). There were no safety concerns. CONCLUSIONS: Central EGFR and HER2neu stratification by IHC and FISH can be used for further pan-HER strategies. Lapatinib with ECF/X was well tolerated, but did not show clear activity in patients with metastatic GC.

Rectal and anal canal neuroendocrine tumours.

Neuroendocrine tumors (NETs) are rare, representing 0.5% of all newly diagnosed malignancies. Rectal and anal canal (AC) NETs account for less than 1% of all rectal and AC cancers. Review our institutional experience on NET of the rectum and AC, with emphasis on demographic, histological and treatment features and oncologic outcomes. The study group was identified from the Portuguese Regional South Oncological Registry. From 2000 to 2014, 22 patients with rectal or AC NETs were treated at our institution. Medical records were retrospectively reviewed. There were 12 males (54.5%) and 10 females (45.5%) and the median age at diagnosis was 59.5 years. The majority had rectal NET (81.8%). All 4 patients with AC NETs had neuroendocrine carcinoid (NEC) tumors. Of the patients with rectal NETs, 3 had NEC and 15 had NET, mainly G1. Different approaches to treatment were made according to histological and staging features. After an average follow-up of 39.1 months, 16 patients were alive and only one with evidence of disease. The median time to progression was 12.4 months and the liver was the most frequent site of metastasis. The European and North American Neuroendocrine Societies offer guidelines for the treatment of rectal NETs. However, for AC NETs there are only small series and not prospective studies due to their rarity, hence the importance to report institutional experience. Our practice demonstrated that primary excisional treatment, regardless the histology, provides a favorable prognosis and long survival.

Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.

siRNA-cell-penetrating peptides complexes as a combinatorial therapy against chronic myeloid leukemia using BV173 cell line as model.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell-penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer-related signaling processes. Both pepM and pepR penetrate the positive Bcr-Abl+ Cell Line (BV173). Five in silico designed anti-Bcr-Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr-Abl downregulation kinetics (48h to 168h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome-wide microarray analysis and further validated by testing BV173 cell cycle and cell proliferation monitoring different genes involved in housekeeping/cell stress (RPL13A, HPRT1), cell proliferation (ki67), cell apoptosis (Caspase 3 and Caspase 9) and cell cycle steps (CDK2, CCDN2, CDKN1A). Assays with a commercial transfection agent were carried out for comparison purposes. Maximal Bcr-Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120h. Both pepM and pepR showed downregulation effects on proliferative CML-related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules. With this work we show the therapeutic potential of a CPP shuttle that combines intrinsic anticancer properties with the ability to deliver functional siRNA into CML cell models. By such combination, the pepM-siRNA conjugates lowered Bcr-Abl gene expression levels more extensively than conventional siRNA delivery technologies and perturbed leukemogenic cell homeostasis, hence revealing their potential as novel alternative scaffolds for CML therapy.

Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles.

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.

Rectal cancer management in elderly patients: experience of a single Portuguese institution.

BACKGROUND: The incidence of rectal cancer increases with age, and older patients are more likely to have other chronic conditions that can affect outcome and tolerability of treatment. OBJECTIVE: The incidence of rectal cancer increases with age, and older patients are more likely to have other chronic conditions that can affect outcome and tolerability of treatment. METHODS: 59 patients aged 75 years and older with stage II-III rectal cancer who were treated during a 3-year period were included in the study. Comorbidities were assessed using the Charlson Comorbidity Index (CCI) and the patients were divided into 2 groups based on their CCI scores: Fit (score of 0-1 points) and Vulnerable (score of greater than or = 2). Primary endpoint was survival at 1 and 3 years. RESULTS: The sample included 43 patients (72.9%) in the Fit group and 16 patients (27.1%) in the Vulnerable group. The most common comorbidities were myocardial infarction, diabetes, and chronic lung disease. One-year survival the same between the groups (P = .330), but 3-year survival was lower in the Vulnerable group patients (83.7% vs 56.3%, respectively; P = .040). The rates of neoadjuvant chemo- and radiotherapy use and low anterior resection performance were the same between the groups. Colostomy closure was achieved more frequently in the Fit group compared with the Vulnerable group (83.3% vs 55.6%; P = .083). There was no difference in mean disease-free survival, grade 3-4 toxicity, and dose reduction between the groups. CONCLUSIONS: Comorbidity assessment should always be included in standard oncological management of elderly patients. Fit patients can be managed with standard treatment and may bene¦t from a conventional, more aggressive approach in their therapy.

Rethinking the capsid proteins of enveloped viruses: multifunctionality from genome packaging to genome transfection.

Regardless of the debate on whether there is a place for viruses in the tree of life, it is consensual that they co-evolve with their hosts under the pressure of genome minimization. The abundance of multifunctional viral structural proteins is a consequence of this pressure. The molecular key to multifunctionality is the existence of intrinsically disordered domains together with ordered domains in the same protein. Capsid proteins, the hallmark of viruses, are not exceptions because they have coexisting ordered and disordered domains that are crucial for multifunctionality. It is also frequent to find supercharged proteins (i.e. proteins for which the net charge per unit molecular mass is > +0.75/kDa) among viral capsid proteins. All flaviviruses having annotated proteins in the ExPASy Viralzone database have supercharged capsid proteins. Moreover, cell-penetrating sequences/domains are frequent in viral proteins, even when they are not supercharged. Altogether, the findings strongly suggest that the ability to translocate membranes was acquired, conserved and optimized throughout the evolution of some viral proteins as part of their multifunctionality. The fitness of capsid proteins to translocate membranes carrying genomes was experimentally demonstrated with dengue virus capsid protein. This protein is potentially able to help the fusion process and translocate the RNA genome across the hemifused membrane formed by the viral envelope and the endosomal membrane. In addition, one of the cell-penetrating domains of the capsid protein also has antibacterial activity. This may be reminiscent of parasitic bacteria-bacteria competition for the same host and shed light on the origins of enveloped viruses.

Shifting gear in antimicrobial and anticancer peptides biophysical studies: from vesicles to cells.

Despite the intensive study on the mechanism of action of membrane-active molecules such as antimicrobial and anticancer peptides, most of the biophysical work has been performed using artificial model systems, mainly lipid vesicles. The use of these systems allows full control of the experimental parameters, and to obtain molecular-level detail on the action of peptides, the correlation with biological action is intangible. Recently, several biophysical methodologies have been translated to studies using bacterial and cancer cells. Here, we review biophysical studies on the mechanism of action of antimicrobial and anticancer peptides performed directly on cells. The data in these studies allow to correlate vesicle-based and cell-based studies and fill the vesicle-cell interdisciplinary gap.

Apoptotic human neutrophil peptide-1 anti-tumor activity revealed by cellular biomechanics.

Cancer remains a major cause of morbidity and mortality worldwide. Although progress has been made regarding chemotherapeutic agents, new therapies that combine increased selectivity and efficacy with low resistance are still needed. In the search for new anticancer agents, therapies based on biologically active peptides, in particular, antimicrobial peptides (AMPs), have attracted attention for their decreased resistance development and low cytotoxicity. Many AMPs have proved to be tumoricidal agents against human cancer cells, but their mode of action is still controversial. The existence of common properties shared by the membranes of bacteria and tumor cells points to similar lipid-targeting mechanisms in both cases. On the other hand, anticancer peptides (ACPs) also induce apoptosis and inhibit angiogenesis. Human neutrophil peptide-1 (HNP-1) is an endogenous AMP that has been implicated in different cellular phenomena such as tumor proliferation. The presence of HNP-1 in the serum/plasma of oncologic patients turns this peptide into a potential tumor biomarker. The present work reveals the different effects of HNP-1 on the biophysical and nanomechanical properties of solid and hematological tumor cells. Studies on cellular morphology, cellular stiffness, and membrane ultrastructure and charge using atomic force microscopy (AFM) and zeta potential measurements show a preferential binding of HNP-1 to solid tumor cells from human prostate adenocarcinoma when compared to human leukemia cells. AFM also reveals induction of apoptosis with cellular membrane defects at very low peptide concentrations. Understanding ACPs mode(s) of action will certainly open innovative pathways for drug development in cancer treatment.