Current Advances in Photodynamic Therapy (PDT) and the Future Potential of PDT-Combinatorial Cancer Therapies.

Photodynamic therapy (PDT) is a two-stage treatment that implies the use of light energy, oxygen, and light-activated compounds (photosensitizers) to elicit cancerous and precancerous cell death after light activation (phototoxicity). The biophysical, bioengineering aspects and its combinations with other strategies are highlighted in this review, both conceptually and as they are currently applied clinically. We further explore the recent advancements of PDT with the use of nanotechnology, including quantum dots as innovative photosensitizers or energy donors as well as the combination of PDT with radiotherapy and immunotherapy as future promising cancer treatments. Finally, we emphasize the potential significance of organoids as physiologically relevant models for PDT.

Associations of Mutually Exclusive Categories of Physical Activity and Sedentary Time With Metabolic Syndrome in Older Adults: An Isotemporal Substitution Approach.

The aim of this study was to examine, theoretically, how reallocating time between the intensity of mutually exclusive categories of physical activity and sedentary behavior time is associated with metabolic syndrome. Four hundred and six older adults (61.6% women) from the second wave of the EpiFloripa Aging Cohort Study were included in the study (mean age 71.7 ± 5.9 years). Isotemporal substitution analysis showed a decrease of 35% (odds ratio: 0.65; 95% confidence interval [0.45, 0.96]) in the risk for metabolic syndrome when replacing 30 min/day of sedentary behavior with an equivalent amount of moderate to vigorous physical activity. Furthermore, it has been observed that older adults classified as low sedentary behavior and physically active were 57% less likely to have metabolic syndrome than participants classified as high sedentary and physically inactive (odds ratio: 0.43; 95% confidence interval [0.19, 0.97]). This study highlights the importance of behavioral categories that may emerge concerning the interrelationships of physical activity and health in older adults, having important implications for future health intervention programs.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

Construction and validation of a regulatory network for pluripotency and self-renewal of mouse embryonic stem cells.

A 30-node signed and directed network responsible for self-renewal and pluripotency of mouse embryonic stem cells (mESCs) was extracted from several ChIP-Seq and knockdown followed by expression prior studies. The underlying regulatory logic among network components was then learned using the initial network topology and single cell gene expression measurements from mESCs cultured in serum/LIF or serum-free 2i/LIF conditions. Comparing the learned network regulatory logic derived from cells cultured in serum/LIF vs. 2i/LIF revealed differential roles for Nanog, Oct4/Pou5f1, Sox2, Esrrb and Tcf3. Overall, gene expression in the serum/LIF condition was more variable than in the 2i/LIF but mostly consistent across the two conditions. Expression levels for most genes in single cells were bimodal across the entire population and this motivated a Boolean modeling approach. In silico predictions derived from removal of nodes from the Boolean dynamical model were validated with experimental single and combinatorial RNA interference (RNAi) knockdowns of selected network components. Quantitative post-RNAi expression level measurements of remaining network components showed good agreement with the in silico predictions. Computational removal of nodes from the Boolean network model was also used to predict lineage specification outcomes. In summary, data integration, modeling, and targeted experiments were used to improve our understanding of the regulatory topology that controls mESC fate decisions as well as to develop robust directed lineage specification protocols.

Patterning pluripotency in embryonic stem cells.

Developmental gradients of morphogens and the formation of boundaries guide the choices between self-renewal and differentiation in stem cells. Still, surprisingly little is known about gene expression signatures of differentiating stem cells at the boundaries between regions. We thus combined inducible gene expression with a microfluidic technology to pattern gene expression in murine embryonic stem cells. Regional depletion of the Nanog transcriptional regulator was achieved through the exposure of cells to microfluidic gradients of morphogens. In this way, we established pluripotency-differentiation boundaries between Nanog expressing cells (pluripotency zone) and Nanog suppressed cells (early differentiation zone) within the same cell population, with a gradient of Nanog expression across the individual cell colonies, to serve as a mimic of the developmental process. Using this system, we identified strong interactions between Nanog and its target genes by constructing a network with Nanog as the root and the measured levels of gene expression in each region. Gene expression patterns at the pluripotency-differentiation boundaries recreated in vitro were similar to those in the developing blastocyst. This approach to the study of cellular commitment at the boundaries between gene expression domains, a phenomenon critical for understanding of early development, has potential to benefit fundamental research of stem cells and their application in regenerative medicine.

The impact of a biocellulose-based repair of fetal open spina bifida on the need to untether the cord: is it time to unify techniques for prenatal repair?

OBJECTIVE: To report the need for cord untethering after prenatal repair of open spina bifida using a unique biocellulose-based technique performed at a later gestational age. METHODS: An observational cohort study was conducted to determine the incidence of tethered cord syndrome. Between May 2013 and May 2022, we performed 172 procedures using the percutaneous fetoscopic approach in fetuses at 26-28 weeks of gestation. After placode dissection, a biocellulose patch was placed to cover the placode, a myofascial flap (when possible) was dissected, and the skin was closed. Owing to death or loss to follow-up, 23 cases were excluded. Cord tethering syndrome was defined as symptoms of medullary stretching, and the infants were evaluated and operated on by local neurosurgeons after an magnetic resonance imaging examination. Infants over 30-month had ambulation and neurodevelopment evaluations (PEDI scale). RESULTS: Among 172 cases operated at a median gestational age of 26.7 weeks and delivered at 33.2 weeks, 149 cases were available for postnatal follow-up, and cord untethering was needed in 4.4% of cases (6/136; excluding 13 cases younger than 12 months). Cerebrospinal fluid diversion and bladder catheterization were needed in 38% and 36% of cases, respectively. Of the 78 cases evaluated at 30 months, 49% were ambulating independently, and 94% had normal social function. CONCLUSION: The biocellulose-based technique was associated with a low rate of cord tethering, wich may be attributed to the lack of the duramater suture during prenatal repair, the formation of a neoduramater and/or later gestational age of surgery.

Vaccinia-related kinase 1 (VRK1) is an upstream nucleosomal kinase required for the assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage.

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiation-induced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses.

Prdm16 is a physiologic regulator of hematopoietic stem cells.

Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.

Preclinical Studies with Glioblastoma Brain Organoid Co-Cultures Show Efficient 5-ALA Photodynamic Therapy.

BACKGROUND: The high recurrence of glioblastoma (GB) that occurs adjacent to the resection cavity within two years of diagnosis urges an improvement of therapies oriented to GB local control. Photodynamic therapy (PDT) has been proposed to cleanse infiltrating tumor cells from parenchyma to ameliorate short long-term progression-free survival. We examined 5-aminolevulinic acid (5-ALA)-mediated PDT effects as therapeutical treatment and determined optimal conditions for PDT efficacy without causing phototoxic injury to the normal brain tissue. METHODS: We used a platform of Glioma Initiation Cells (GICs) infiltrating cerebral organoids with two different glioblastoma cells, GIC7 and PG88. We measured GICs-5-ALA uptake and PDT/5-ALA activity in dose-response curves and the efficacy of the treatment by measuring proliferative activity and apoptosis. RESULTS: 5-ALA (50 and 100 µg/mL) was applied, and the release of protoporphyrin IX (PpIX) fluorescence measures demonstrated that the emission of PpIX increases progressively until its stabilization at 24 h. Moreover, decreased proliferation and increased apoptosis corroborated the effect of 5-ALA/PDT on cancer cells without altering normal cells. CONCLUSIONS: We provide evidence about the effectiveness of PDT to treat high proliferative GB cells in a complex in vitro system, which combines normal and cancer cells and is a useful tool to standardize new strategic therapies.

Comprehensive analysis of lymph node stroma-expressed Ig superfamily members reveals redundant and nonredundant roles for ICAM-1, ICAM-2, and VCAM-1 in lymphocyte homing.

Although it is well established that stromal intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule-1 (VCAM-1) mediate lymphocyte recruitment into peripheral lymph nodes (PLNs), their precise contributions to the individual steps of the lymphocyte homing cascade are not known. Here, we provide in vivo evidence for a selective function for ICAM-1 > ICAM-2 > VCAM-1 in lymphocyte arrest within noninflamed PLN microvessels. Blocking all 3 CAMs completely inhibited lymphocyte adhesion within PLN high endothelial venules (HEVs). Post-arrest extravasation of T cells was a 3-step process, with optional ICAM-1-dependent intraluminal crawling followed by rapid ICAM-1- or ICAM-2-independent diapedesis and perivascular trapping. Parenchymal motility of lymphocytes was modestly reduced in the absence of ICAM-1, while ICAM-2 and alpha4-integrin ligands were not required for B-cell motility within follicles. Our findings highlight nonredundant functions for stromal Ig family CAMs in shear-resistant lymphocyte adhesion in steady-state HEVs, a unique role for ICAM-1 in intraluminal lymphocyte crawling but redundant roles for ICAM-1 and ICAM-2 in lymphocyte diapedesis and interstitial motility.

Understanding the Molecular Basis of iPSC Reprogrammed Cells to Fulfil Their Expectations in Future Clinical Applications.

IPSC-based disease modelling and pluripotency studies have sparked widespread enthusiasm for more than 16 years of research [...].

CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs.

The realization of the full potential of human pluripotent stem cells (hPSCs), including human induced PSCs (iPSC), relies on the ability to precisely edit their genome in a locus-specific and multiplex manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) serve as a guide for the endonuclease Cas9 (CRISPR-associated protein 9) to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. CRISPR/Cas9-mediated editing has become the gold standard for precise genome manipulation as it offers a unique, versatile, and limitless tool for fast, robust, and efficient genome editing. Here, we provide a protocol to successfully generate gene knockout and/or knockin iPSCs. We include detailed information on the design of guide RNAs (gRNAs), T7 endonuclease assay to detect on-target CRISPR/Cas9 editing events, DNA electroporation of the iPSCs with a ribonucleoprotein complex, and single-cell cloning steps for the selection of the genome-edited iPSC clones.

Esrrb Regulates Specific Feed-Forward Loops to Transit From Pluripotency Into Early Stages of Differentiation.

Characterization of pluripotent states, in which cells can both self-renew or differentiate, with the irreversible loss of pluripotency, are important research areas in developmental biology. Although microRNAs (miRNAs) have been shown to play a relevant role in cellular differentiation, the role of miRNAs integrated into gene regulatory networks and its dynamic changes during these early stages of embryonic stem cell (ESC) differentiation remain elusive. Here we describe the dynamic transcriptional regulatory circuitry of stem cells that incorporate protein-coding and miRNA genes based on miRNA array expression and quantitative sequencing of short transcripts upon the downregulation of the Estrogen Related Receptor Beta (Esrrb). The data reveals how Esrrb, a key stem cell transcription factor, regulates a specific stem cell miRNA expression program and integrates dynamic changes of feed-forward loops contributing to the early stages of cell differentiation upon its downregulation. Together these findings provide new insights on the architecture of the combined transcriptional post-transcriptional regulatory network in embryonic stem cells.

ABO gene editing for the conversion of blood type A to universal type O in Rhnull donor-derived human-induced pluripotent stem cells.

The limited availability of red cells with extremely rare blood group phenotypes is one of the global challenges in transfusion medicine that has prompted the search for alternative self-renewable pluripotent cell sources for the in vitro generation of red cells with rare blood group types. One such phenotype is the Rhnull , which lacks all the Rh antigens on the red cell membrane and represents one of the rarest blood types in the world with only a few active blood donors available worldwide. Rhnull red cells are critical for the transfusion of immunized patients carrying the same phenotype, besides its utility in the diagnosis of Rh alloimmunization when a high-prevalence Rh specificity is suspected in a patient or a pregnant woman. In both scenarios, the potential use of human-induced pluripotent stem cell (hiPSC)-derived Rhnull red cells is also dependent on ABO compatibility. Here, we present a CRISPR/Cas9-mediated ABO gene edition strategy for the conversion of blood type A to universal type O, which we have applied to an Rhnull donor-derived hiPSC line, originally carrying blood group A. This work provides a paradigmatic example of an approach potentially applicable to other hiPSC lines derived from rare blood donors not carrying blood type O.

Diagnostic accuracy of regadenoson stress echocardiography: concordance with gated-spect myocardial perfusion imaging.

Regadenoson Stress Echocardiography (RSE) can detect myocardial ischemia, and its diagnostic accuracy should be evaluated. We sought to investigate the agreement between RSE and gated-SPECT myocardial perfusion imaging (MPI) and appraise its diagnostic accuracy. Consecutive patients (n = 202) referred for non-invasive evaluation of myocardial ischemia, with (38.6%) or without a previous coronary artery disease (CAD) diagnosis, were enrolled. Both tests were performed simultaneously. Invasive coronary angiography (CA) is considered the gold standard. The mean age was 70.9 (9.8) years, and 59.9% were male. The prevalence of cardiovascular risk factors (arterial hypertension [81.7%], diabetes mellitus [37.6%], hypercholesterolemia [71.8%], and smoking [18.8%]) was high. Forty-four patients (21.8%) had a non-interpretable electrocardiogram, 15 (34.1%) of them were a result of ventricular paced-rhythm, while 29 (65.9%) were a result of advanced left ventricular branch block. The overall agreement between both diagnostic techniques was good: Gwet's AC1 0.66 (CI95% 0.55 to 0.76), and it was higher in patients without a previous CAD diagnosis: 0.76 (CI95% 0.65 to 0.87). In the biased sample (those who underwent CA), RSE and nuclear study sensitivity was 0.50 and 0.78 and specificity was 0.75 and 0.75, respectively. We noted a dramatic reduction in sensitivity for RSE after debiasing (debiased sensitivity of 0.16), and the negative predictive value was similar to the biased and debiased samples. RSE is in strong agreement with gated-SPECT MPI. However, its low sensitivity and negative predictive value preclude its use as a bedside test to detect myocardial ischemia.

An Esrrb and Nanog Cell Fate Regulatory Module Controlled by Feed Forward Loop Interactions.

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.

The "Controversial Cundurango Cure": Medical professionalization and the global circulation of drugs.

This article examines the medical and political discussions regarding a controversial medicinal bark from Ecuador - cundurango - that was actively sponsored by the Ecuadorian government as a new botanical cure for cancer in the late nineteenth century United States and elsewhere. The article focuses on the commercial and diplomatic interests behind the public discussion and advertising techniques of this drug. It argues that diverse elements - including the struggle for positioning scientific societies and the disapproval of the capacities of Ecuadorian doctors, US abolitionist history, regional and local political struggles - played a role in the quackery accusations against cundurango and its promoters. The development and international trade of this remedy offer interesting insights into the global history of drugs, particularly how medical knowledge was challenged during a period when scientific medicine was struggling for hegemony. It explores how newspapers expanded "the public interest" in a possible cancer cure.

Glia Crosstalk in Neuroinflammatory Diseases.

Neuroinflammation constitutes a fundamental cellular process to signal the loss of brain homeostasis. Glial cells play a central role in orchestrating these neuroinflammation processes in both deleterious and beneficial ways. These cellular responses depend on their intercellular interactions with neurons, astrocytes, the blood-brain barrier (BBB), and infiltrated T cells in the central nervous system (CNS). However, this intercellular crosstalk seems to be activated by specific stimuli for each different neurological scenario. This review summarizes key studies linking neuroinflammation with certain neurodegenerative diseases such as Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) and for the development of better therapeutic strategies based on immunomodulation.

Need to promote healthy lifestyle as primary prevention to the COVID-19 and to improve the immune response to vaccines.

Letter to the editor.