The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation.

Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.

Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Bemarituzumab as first-line treatment for locally advanced or metastatic gastric/gastroesophageal junction adenocarcinoma: final analysis of the randomized phase 2 FIGHT trial.

BACKGROUND: We report the final results of the randomized phase 2 FIGHT trial that evaluated bemarituzumab, a humanized monoclonal antibody selective for fibroblast growth factor receptor 2b (FGFR2b), plus mFOLFOX6 in patients with FGFR2b-positive (2 + /3 + membranous staining by immunohistochemistry), HER-2-negative gastric or gastroesophageal junction cancer (GC). METHODS: Patients received bemarituzumab (15 mg/kg) or placebo once every 2 weeks with an additional bemarituzumab (7.5 mg/kg) or placebo dose on cycle 1 day 8. All patients received mFOLFOX6. The primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included overall survival (OS), objective response rate, and safety. Efficacy was evaluated after a minimum follow-up of 24 months. RESULTS: In the bemarituzumab-mFOLFOX6 (N = 77) and placebo-mFOLFOX6 (N = 78) arms, respectively, 59.7% and 66.7% of patients were FGFR2b-positive in ≥ 10% of tumor cells. The median PFS (95% confidence interval [CI]) was 9.5 months (7.3-13.7) with bemarituzumab-mFOLFOX6 and 7.4 months (5.7-8.4) with placebo-mFOLFOX6 (hazard ratio [HR], 0.72; 95% CI 0.49-1.08); median OS (95% CI) was 19.2 (13.6-24.2) and 13.5 (9.3-15.9) months, respectively (HR 0.77; 95% CI 0.52-1.14). Observed efficacy in FGFR2b-positive GC in ≥ 10% of tumor cells was: PFS: HR 0.43 (95% CI 0.26-0.73); OS: HR 0.52 (95% CI 0.31-0.85). No new safety findings were reported. CONCLUSIONS: In FGFR2b-positive advanced GC, the combination of bemarituzumab-mFOLFOX6 led to numerically longer median PFS and OS compared with mFOLFOX6 alone. Efficacy was more pronounced with FGFR2b overexpression in ≥ 10% of tumor cells. Confirmatory phase 3 trials are ongoing (NCT05052801, NCT05111626). CLINICAL TRIAL REGISTRATION: NCT03694522.

Characterization and Vaccine Potential of Outer Membrane Vesicles from Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen.

Fluctuating asymmetry in third molar agenesis as an aid to estimate socioeconomic status.

Traditionally, dental identification techniques are used to establish identity or assist in reconstructing an individual's biological profile. However, other aspects of identity, namely socioeconomic status (SES), can be estimated through teeth. This work aims to evaluate the influence of SES on third molar agenesis in a Portuguese population. X-rays from 448 subjects (223 belonging to a high and 225 to a low socioeconomic status) were assessed and demographic data (age, sex) and dental history were registered. Frequencies and associations between the variables were analyzed using the chi-square test. For each group, differences between third molar agenesis were studied using the Wilcoxon test. The significance level was 5%. X-rays displaying at least one agenesis were more common in females (in both SES groups). Differences between socioeconomic status were found in female subjects' upper right and lower left third molars, with a higher frequency of agenesis in the lower SES group. Agenesis of lower third molars displayed fluctuant asymmetry in both groups, whereas agenesis of upper third molars was also present in the lower SES group. These results suggest that socioeconomic status can affect third molar agenesis prevalence, and fluctuant asymmetry seems more prevalent in the lower SES, as it affects all third molars.

Bemarituzumab in patients with FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma (FIGHT): a randomised, double-blind, placebo-controlled, phase 2 study.

BACKGROUND: Outcomes are poor in patients with HER2-negative, advanced gastric or gastro-oesophageal junction adenocarcinomas. In this study, we investigated efficacy and safety of the first-in-class, afucosylated, humanised IgG1 anti-fibroblast growth factor receptor 2 isoform IIb (FGFR2b) monoclonal antibody bemarituzumab with modified 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) in patients with FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma. METHODS: In the randomised, double-blind, placebo-controlled phase 2 trial (FIGHT), patients aged 18 years and older with HER2 non-positive, FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma, and an Eastern Cooperative Oncology Group performance status of 0-1 were recruited from 144 clinical sites across 17 countries. Patients with previous treatment with any selective inhibitor of the FGF-FGFR pathway were excluded. Eligible patients were randomly assigned (1:1), using permuted-block randomisation (block size of four) and a central interactive voice-web-based response system, stratified by geographical region, previous treatment with curative intent, and administration of mFOLFOX6 while being screened for FGFR2b status, to either bemarituzumab (15 mg/kg of bodyweight) or matched placebo intravenously every 2 weeks. All patients also received mFOLFOX6 (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, and 5-fluorouracil as a 400 mg/m2 bolus followed by 2400 mg/m2 over approximately 46 h) intravenously every 2 weeks. Patients were given treatment until disease progression (defined by Response Evaluation Criteria in Solid Tumours [RECIST] version 1.1), unacceptable toxicity, withdrawal of consent, or death. The primary endpoint was progression-free survival in the intention-to-treat population (defined as all patients randomly assigned to treatment). Safety was assessed in all patients who received at least one dose of assigned treatment. This study is registered with ClinicalTrials.gov, NCT03694522, and is now complete. FINDINGS: Between Nov 14, 2017, and May 8, 2020, 910 patients were screened and 155 were randomly assigned to the bemarituzumab (n=77) or placebo group (n=78). Median age was 60·0 years (IQR 51·0-67·0), 44 (28%) participants were women, 111 (72%) were men, 89 (57%) were Asian, and 61 (39%) were White. At the time of the primary analysis and at a median follow-up of 10·9 months (IQR 6·3-14·2), median progression-free survival was 9·5 months (95% CI 7·3-12·9) in the bemarituzumab group and 7·4 months (5·8-8·4) in the placebo group (hazard ratio [HR] 0·68 [95% CI 0·44-1·04; p=0·073). Common grade 3 or worse adverse events were decreased neutrophil count (23 [30%] of 76 in the bemarituzumab group vs 27 [35%] of 77 in the placebo group), cornea disorder (18 [24%] vs none), neutropenia (ten [13%] vs seven [9%]), stomatitis (seven [9%] vs one [1%]), and anaemia (six [8%] vs ten [13%]). Serious treatment-emergent adverse events were reported in 24 (32%) patients in the bemarituzumab group and 28 (36%) in the placebo group. Serious mFOLFOX6 treatment-related adverse events occurred in nine (12%) patients in the bemarituzumab group and in 15 (19%) patients in the placebo group. All-grade corneal events (adverse events of special interest) occurred in 51 (67%) patients in the bemarituzumab group and eight (10%) in the placebo group; grade 3 corneal events were reported only in 18 (24%) patients in the bemarituzumab group. Treatment-related deaths occurred in three patients in the bemarituzumab group (two due to sepsis, one due to pneumonia) and none in the placebo group. INTERPRETATION: In this exploratory phase 2 study, despite no statistically significant improvement in progression-free survival, treatment with bemarituzumab showed promising clinical efficacy. Confirmatory phase 3 trials of bemarituzumab plus mFOLFOX6 powered to demonstrate statistical significance are being investigated in patients with previously untreated, FGFR2b-overexpressing, advanced gastric or gastro-oesophageal junction adenocarcinoma. FUNDING: Five Prime Therapeutics.

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

In Vitro Evaluation of Lipopolyplexes for Gene Transfection: Comparing 2D, 3D and Microdroplet-Enabled Cell Culture.

Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.

Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient-sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB-4, HL-60 and KG-1) and their impact on autophagy and survival was characterized. Data show that whereas KG-1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co-activation of AMPK and mTORC1 associated with increased autophagy, NB-4 and HL-60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG-1 cells' survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti-leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents.

Gla-rich protein acts as a calcification inhibitor in the human cardiovascular system.

OBJECTIVE: Vascular and valvular calcifications are pathological processes regulated by resident cells, and depending on a complex interplay between calcification promoters and inhibitors, resembling skeletal metabolism. Here, we study the role of the vitamin K-dependent Gla-rich protein (GRP) in vascular and valvular calcification processes. APPROACH AND RESULTS: Immunohistochemistry and quantitative polymerase chain reaction showed that GRP expression and accumulation are upregulated with calcification simultaneously with osteocalcin and matrix Gla protein (MGP). Using conformation-specific antibodies, both γ-carboxylated GRP and undercarboxylated GRP species were found accumulated at the sites of mineral deposits, whereas undercarboxylated GRP was predominant in calcified aortic valve disease valvular interstitial cells. Mineral-bound GRP, MGP, and fetuin-A were identified by mass spectrometry. Using an ex vivo model of vascular calcification, γ-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through α-smooth muscle actin upregulation and osteopontin downregulation. Immunoprecipitation assays showed that GRP is part of an MGP-fetuin-A complex at the sites of valvular calcification. Moreover, extracellular vesicles released from normal vascular smooth muscle cells are loaded with GRP, MGP, and fetuin-A, whereas under calcifying conditions, released extracellular vesicles show increased calcium loading and GRP and MGP depletion. CONCLUSIONS: GRP is an inhibitor of vascular and valvular calcification involved in calcium homeostasis. Its function might be associated with prevention of calcium-induced signaling pathways and direct mineral binding to inhibit crystal formation/maturation. Our data show that GRP is a new player in mineralization competence of extracellular vesicles possibly associated with the fetuin-A-MGP calcification inhibitory system. GRP activity was found to be dependent on its γ-carboxylation status, with potential clinical relevance.

Change, stability and prediction of gross motor co-ordination in Portuguese children.

BACKGROUND: The knowledge about intra- and inter-individual variation can stimulate attempts at description, interpretation and prediction of motor co-ordination (MC). AIM: To analyse change, stability and prediction of motor co-ordination (MC) in children. SUBJECTS AND METHODS: A total of 158 children, 83 boys and 75 girls, aged 6, 7 and 8 years, were evaluated in 2006 and re-evaluated in 2012 at 12, 13 and 14 years of age. MC was assessed through the Kiphard-Schilling's body co-ordination test and growth, skeletal maturity, physical fitness, fundamental motor skills (FMS), physical activity and socioeconomic status (SES) were measured and/or estimated. RESULTS: Repeated-measures MANOVA indicated that there was a significant effect of group, sex and time on a linear combination of the MC tests. Univariate tests revealed that group 3 (8-14 years) scored significantly better than group 1 (6-12 years) in all MC tests and boys performed better than girls in hopping for height and moving sideways. Scores in MC were also higher at follow-up than at baseline. Inter-age correlations for MC were between 0.15-0.74. Childhood predictors of MC were growth, physical fitness, FMS, physical activity and SES. Biological maturation did not contribute to prediction of MC. CONCLUSION: MC seemed moderately stable from childhood through adolescence and, additionally, inter-individual predictors at adolescence were growth, FMS, physical fitness, physical activity and SES.

Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation.

How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization.We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation.

Multielectrocatalysis by layer-by-layer films based on pararosaniline and vanadium-substituted phosphomolybdate.

Hybrid multilayer films based on the two molecular species pararosaniline (PR) and Keggin-type polyoxometalate K5[PMo11VO40)] (PMo11V) were prepared on different substrates using the electrostatic layer-by-layer (LbL) self-assembly method. The film buildup, monitored by electronic spectroscopy, showed a regular stepwise growth, and X-ray photoelectron spectroscopy data confirmed the presence of both molecular components within the LbL films. Scanning electron microscopy images revealed a completely covered surface with a nonuniform distribution of film components, and atomic force microscopy images confirmed a rough surface. The film electrochemical responses and permeability were studied by cyclic voltammetry. Films revealed three Mo-based redox processes (Mo(VI) → Mo(V)) and one V-based redox process (V(V) → V(IV)) in the potential range between 0.8 and -0.4 V vs Ag/AgCl. Studies with the redox probes [Fe(CN)6](3-/4-) and [Ru(NH3)6](3+/2+) showed that the films maintain the permeability even after six bilayers. Furthermore, the {PR/PMo11V}n multilayer films exhibit excellent Mo-based electrocatalytic activity toward reduction of iodate and V-based electrocatalytic activity toward ascorbic acid oxidation, thus acting as a versatile multielectrocatalyst.

Gross motor coordination and weight status of Portuguese children aged 6-14 years.

OBJECTIVES: To construct age- and gender-specific percentiles for gross motor coordination (MC) tests and to explore differences in gross MC in normal-weight, overweight and obese children. METHODS: Data are from the "Healthy Growth of Madeira Study," a cross-sectional study carried out in children, aged 6-14 years. All 1,276 participants, 619 boys and 657 girls, were assessed for gross MC (Körperkoordinations Test für Kinder, KTK), anthropometry (height and body mass), physical activity (Baecke questionnaire) and socioeconomic status (SES). Centile curves for gross MC were obtained for boys and girls separately using generalized additive models for location, scale and shape. RESULTS: A significant main effect for age was found in walking backwards and moving sideways. Boys performed significantly better than girls on moving sideways. At the upper limit of the distributions, interindividual variability was higher in hopping on one leg (girls) and jumping and moving sideways (boys and girls). One-way ANCOVA, controlling for age, physical activity and SES, indicated that normal-weight children scored significantly better than their obese peers in all gross MC tests. Overweight boys and girls also scored significantly better than their obese colleagues in some MC tests. CONCLUSIONS: These centile curves can be used as reference data in Portuguese children and youth, aged 6-14 years. Being overweight or obese was a major limitation in MC tests and, therefore, of the children's health- and performance-related physical fitness.

Marital status and abortion among young women in Rupandehi, Nepal.

BACKGROUND: Despite liberalization of the Nepal abortion law, young women continue to experience barriers to safe abortion services. We hypothesize that marital status may differentially impact such barriers, given the societal context of Nepal. METHODS: We evaluated differences in reproductive knowledge and attitudes by marital status with a probability-based, cross-sectional survey of young women in Rupandehi district, Nepal. Participants (N = 600) were surveyed in 2012 on demographics, romantic experiences, media habits, reproductive information, and abortion knowledge and attitudes. We used logistic regression to assess differences by marital status, controlling for age. RESULTS: Participants, who comprised never-married (54%) and ever-married women (45%), reported good access to basic reproductive health and abortion information. Social desirability bias might have prevented reporting of premarital romantic and sexual activity given that participants reported more premarital activities for their friends than for themselves. Only 45% knew that abortion was legal, and fewer ever-married women were aware of abortion legality. Never-married women expected more negative responses from having an abortion than ever-married women. CONCLUSIONS: Findings highlight the need for providing sexual and reproductive health care information and services to young women regardless of marital status.

A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants.

The aim of this study was to propose and validate a new unified method for testing dissolution rates of bioactive glasses and their variants, and the formation of calcium phosphate layer formation on their surface, which is an indicator of bioactivity. At present, comparison in the literature is difficult as many groups use different testing protocols. An ISO standard covers the use of simulated body fluid on standard shape materials but it does not take into account that bioactive glasses can have very different specific surface areas, as for glass powders. Validation of the proposed modified test was through round robin testing and comparison to the ISO standard where appropriate. The proposed test uses fixed mass per solution volume ratio and agitated solution. The round robin study showed differences in hydroxyapatite nucleation on glasses of different composition and between glasses of the same composition but different particle size. The results were reproducible between research facilities. Researchers should use this method when testing new glasses, or their variants, to enable comparison between the literature in the future.

The radiographic visibility of the root pulp of the third lower molar as an age marker.

PURPOSE: The aim of this work was to determine the usefulness of the visibility of the dental pulp in lower third molars in forensic age estimation. METHODS: Dental pulp visibility on lower third molars was assessed using a sample of 487 orthopantomograms. Intra and inter-observer agreement was determined using the Cohen's kappa test. A descriptive analysis of the stages according to age was done. The relationship between age and stage attainment was assessed using Chi square test and the strength and direction of the linear relationship between pulp visualization stage and chronological age was evaluated using Spearman rank order correlation (rho). Equations for predicting an age above 21 years were developed using logistic regression. The level of significance was defined at p < 0.05. RESULTS: The relationship between age and stage attainment had statistical significance for both sexes (p < 0.001). There was a medium positive correlation between the two variables for both genders (Spearman ρ = 0.420, p < 0.001 and Spearman ρ = 0.454, p < 0.001, for males and females respectively). The model built for age estimation successfully predicted age over 21 in 96.2% of the females and in 96.9% of the males. However, only 19.6 and 27.0% of predictions were accurate for the group that was younger than 21, for females and males, respectively. CONCLUSIONS: The accuracy of predictions for the group younger than 21 years of age was low, meaning that this methodology may not be suitable for age estimation. Still, stage 3 alone proved to be a suitable age marker for determining an age over 21 years.

Dynamic migration and cell-cell interactions of early reprogramming revealed by high-resolution time-lapse imaging.

Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multicolor time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as 5 minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells that emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies, and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct two-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate that E-cadherin is required for proper cellular interactions from an early stage of reprogramming, including the two-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.

Are deciduous upper molars and lower canines useful for sex estimation?

OBJECTIVE: This paper investigates whether deciduous upper molars and lower canines have sexual dimorphic features, exploring these teeth' dimensions and the presence of Zuckerkandl's tubercle and Carabelli's cusp on the first and second upper molars. DESIGN: We analyzed 64 pairs of dental plaster casts from 34 females and 30 males aged between 3 and 12 years. We measured the first and second deciduous upper molars and the lower deciduous canines (maximum mesiodistal and buccolingual length), and we registered the presence of the Zuckerkandl's tubercle and the Carabelli's cusp on the first and second upper molars, respectively. RESULTS: Regarding the differentiation between sexes using Carabelli's cusp and Zuckerkandl's tubercle, the classification was not independent of Carabelli's cusp presence only for tooth 65 (p = 0.035). In all other teeth, whether for Carabelli's cusp or Zuckerkandl's tubercle, their presence was similar for both sexes. There were statistically significant differences between sexes (p < 0.05) for the buccolingual measurements of both upper second molars, the first right upper molar, and the right canine. The developed model allowed for a 64.1% accuracy in sex estimation. CONCLUSIONS: The study suggests that while Carabelli's cusp and Zuckerkandl's tubercle in upper deciduous molars don't consistently differ between sexes, tooth size, particularly the buccolingual measurements of certain teeth, including upper deciduous molars and lower canines, may provide a more reliable criterion for sex estimation. The developed model depicted moderate accuracy, underscoring the need for a multifactorial approach when estimating sex from skeletal remains. It suggests that while dental features can contribute to sex estimation, they should be used in conjunction with other skeletal or molecular markers to improve accuracy.

Silver Nanoparticles (AgNPs) Uptake by Caveolae-Dependent Endocytosis is Responsible for Their Selective Effect Towards Castration Resistant Prostate Cancer.

Purpose: Castration Resistant Prostate Cancer (CRPC) is characterized by poor prognosis and limited therapeutic options. AgNPs functionalized with glucose (G-AgNPs) were observed cytotoxic to CRPC cell lines (PC-3 and Du-145) and not LNCaP. This study aims to evaluate AgNPs and G-AgNPs' uptake mechanisms in these cells and understand their role in the selective effect against CRPC cells. Methods: Uptake of AgNPs and G-AgNPs was assessed through transmission electron microscopy (TEM). A microRNA (miRNAs) analysis approach was used to uncover the main molecular differences responsible for the endocytic mechanisms' regulation. Caveolin (Cav) 1 and 2 mRNA and protein levels were assessed in the three cell lines. Caveolae-dependent endocytosis was inhibited with genistein or siCav1- and siCav2- in PC-3 and Du-145 and resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration. Caveolae-dependent endocytosis was induced with Cav1+ and Cav2+ plasmids in LNCaP, resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration and TEM to assess their location. Results: AgNPs and G-AgNPs were not uptaked by LNCaP. miRNA analysis revealed 37 upregulated and 90 downregulated miRNAs. Functional enrichment analysis of miRNAs' targets resulted in enrichment of terms related to endocytosis and caveolae. We observed that Cav1 and Cav2 are not expressed in LNCaP. Inhibiting caveolae-dependent endocytosis in Du-145 and PC-3 led to a significative reduction of cytotoxic capacity of AgNPs and G-AgNPs and induction of caveolae-dependent endocytosis in LNCaP lead to a significative increase as well as their uptake by cells. Conclusion: This study shows the potential of these AgNPs as a new therapeutic approach directed to CRPC patients, uncovers caveolae-dependent endocytosis as the uptake mechanism of these AgNPs and highlights deregulation of Cav1 and Cav2 expression as a key difference in hormone sensitive and resistant PCa cells which may be responsible for drug resistance.