Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity.

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

Impacts of a Dysphagia Screening Questionnaire on Speech Pathology Input Using a Transdisciplinary Approach for Patients with Chronic Obstructive Pulmonary Disease in a Pulmonary Rehabilitation Program.

Patients with chronic obstructive pulmonary disease (COPD) in pulmonary rehabilitation programs (PRPs) are not routinely screened for dysphagia. An Australian regional health service audit revealed that patients with COPD are frequently referred to speech pathology during acute admissions, rather than proactively to mitigate the risk of dysphagia-related consequences. Referral patterns to speech pathology using a novel transdisciplinary approach for identifying at risk for dysphagia patients in a PRP were explored. The aim of this study was to investigate the impact of a transdisciplinary dysphagia screening questionnaire on speech pathology referrals within a cohort of patients with COPD enrolled in a PRP. This quasi-experimental study introduced a dysphagia screening questionnaire in a PRP using a transdisciplinary approach. A retrospective audit of PRP patients (n = 563) between 01/01/2014 and 31/12/2018 was conducted to identify the frequency of referrals to speech pathology for dysphagia. Data was compared to a cohort of patients (n = 50) enrolled in the PRP (from 01/02/21 to 30/11/21) after introduction of the questionnaire using Fisher's exact test. Less than 1% (n = 4/563) of PRP patients were referred to speech pathology prior to implementation of the questionnaire. Following the implementation, referrals to speech pathology significantly increased to 16% (8/50) (X2 = 7.72, P < 0.05; odds ratio = 7.89 95% CI [1.94, 32.1]). Introducing a dysphagia screening questionnaire increased referrals to speech pathology from a PRP. This study demonstrated the potential for a transdisciplinary approach in early screening for patients at risk of dysphagia for patients with COPD. Further research is encouraged to explore patient motivation towards speech pathology input with COPD-related dysphagia and clinicians' perceived self-efficacy in using the questionnaire.

Parental Education for the Prevention of Plagiocephaly.

INTRODUCTION: The American Academy of Pediatrics Back-to-Sleep Campaign significantly reduced infant mortality from sudden infant death syndrome. As a result of prolonged supine positioning, the incidence of deformational plagiocephaly has also risen 5-fold since its adoption. We aimed to improve the current educational paradigm for new parents with the goal of reducing the incidence of plagiocephaly within the confines of the Back-to-Sleep Campaign. We hypothesized that the early addition of plagiocephaly focused education for parents would reduce cephalic index, the ratio of head width to length, used as an easily measured objective proxy for positional plagiocephaly. METHODS: Children were screened at their newborn visit. Premature newborns and those diagnosed with craniofacial disorders were excluded. For those enrolled, biparietal and anteroposterior measurements of the head were obtained using manual calipers to obtain cephalic index. Subjects randomly assigned to the intervention group were shown a 2-minute video and given an educational pamphlet on methods to prevent plagiocephaly. Unpaired 2-sample t tests comparing mean differences in intervention and control were performed. RESULTS: Thirty-nine subjects were enrolled as of November 2023 with variable lengths of follow-up completed. The average baseline cephalic index for subjects in the control group was 82.7 and 83.8 for intervention group. Unpaired 2-sample t tests were performed at 2-, 4-, and 6-month time points to analyze the difference between groups. At 4 months, average cephalic index for subjects in the control and treatment group, respectively, was 90.6 and 83.4 (P = 0.02). SIGNIFICANCE: Parental education at the newborn visit led to decreases in cephalic index, a proxy for positional plagiocephaly, compared with control patients. This simple intervention has the potential to reduce parental stress and healthcare costs associated with the evaluation and treatment of plagiocephaly.

NF-κB signaling in neoplastic transition from epithelial to mesenchymal phenotype.

NF-κB transcription factors are critical regulators of innate and adaptive immunity and major mediators of inflammatory signaling. The NF-κB signaling is dysregulated in a significant number of cancers and drives malignant transformation through maintenance of constitutive pro-survival signaling and downregulation of apoptosis. Overactive NF-κB signaling results in overexpression of pro-inflammatory cytokines, chemokines and/or growth factors leading to accumulation of proliferative signals together with activation of innate and select adaptive immune cells. This state of chronic inflammation is now thought to be linked to induction of malignant transformation, angiogenesis, metastasis, subversion of adaptive immunity, and therapy resistance. Moreover, accumulating evidence indicates the involvement of NF-κB signaling in induction and maintenance of invasive phenotypes linked to epithelial to mesenchymal transition (EMT) and metastasis. In this review we summarize reported links of NF-κB signaling to sequential steps of transition from epithelial to mesenchymal phenotypes. Understanding the involvement of NF-κB in EMT regulation may contribute to formulating optimized therapeutic strategies in cancer. Video Abstract.

Method for noninvasive whole-body stimulation with spinning oscillating magnetic fields and its safety in mice.

We recently reported shrinkage of untreatable recurrent glioblastoma (GBM) in an end-stage patient using noninvasive brain stimulation with a spinning oscillating magnetic field (sOMF)-generating device called the Oncomagnetic device. Our in vitro experiments demonstrated selective cancer cell death while sparing normal cells by sOMF-induced increase in intracellular reactive oxygen species (ROS) levels due to magnetic perturbation of mitochondrial electron transport. Here, we describe the results of an in vivo study assessing the toxicity of chronic sOMF stimulation in mice using a newly constructed apparatus comprised of the sOMF-generating active components of the Oncomagnetic device. We chronically stimulated 10 normal 60-day old female C57BL/6 mice in their housing cages for 2 h 3 times a day, as in the patient treatment protocol, over 4 months. We also studied the effects of 2-h acute sOMF stimulation. Our observations and those of blinded independent veterinary staff observers, indicated no significant adverse effects of chronic or acute sOMF stimulation on the health, behavior, electrocardiographic and electroencephalographic activities, hematologic profile, and brain and other tissue and organ morphology of treated mice compared to age-matched untreated control mice. These findings suggest that short- and long-term therapies with the Oncomagnetic device are safe and well tolerated.

Patient perceptions of the benefits and barriers of virtual postnatal care: a qualitative study.

OBJECTIVE: The objective of this study is to understand the perceptions of new mothers using virtual care via video conferencing to gain insight into the benefits and barriers of virtual care for obstetric patients. METHODS: Semi-structured interviews were conducted with 15 patients attending the Kingston Health Sciences Centre. The interviews were 20-25 min in length and recorded through an audio recorder. Thematic analysis was conducted in order to derive the major themes explored in this study. RESULTS: New mothers must often adopt new routines to balance their needs and their child's needs. These routines could impact compliance and motivation to attend follow-up care. In our study, participants expressed high satisfaction with virtual care, emphasizing benefits related to comfort, convenience, communication, socioeconomic factors, and the ease of technology use. Participants also perceived that they could receive emotional support and build trust with their health care providers despite the remote nature of their care. Due to its ease of use and increased accessibility, we argue that virtual care shows promise to facilitate long-term compliance to care in obstetric patients. CONCLUSIONS: Virtual care is a useful modality that could improve compliance to obstetric care. Further research and clinical endeavours should examine how social factors and determinants intersect to determine how they underpin patient perceptions of virtual and in-person care.

Functional compensation between hematopoietic stem cell clones in vivo.

In most organ systems, regeneration is a coordinated effort that involves many stem cells, but little is known about whether and how individual stem cells compensate for the differentiation deficiencies of other stem cells. Functional compensation is critically important during disease progression and treatment. Here, we show how individual hematopoietic stem cell (HSC) clones heterogeneously compensate for the lymphopoietic deficiencies of other HSCs in a mouse. This compensation rescues the overall blood supply and influences blood cell types outside of the deficient lineages in distinct patterns. We find that highly differentiating HSC clones expand their cell numbers at specific differentiation stages to compensate for the deficiencies of other HSCs. Some of these clones continue to expand after transplantation into secondary recipients. In addition, lymphopoietic compensation involves gene expression changes in HSCs that are characterized by increased lymphoid priming, decreased myeloid priming, and HSC self-renewal. Our data illustrate how HSC clones coordinate to maintain the overall blood supply. Exploiting the innate compensation capacity of stem cell networks may improve the prognosis and treatment of many diseases.

Multifocal transcranial stimulation in chronic ischemic stroke: A phase 1/2a randomized trial.

BACKGROUND AND PURPOSE: Repetitive transcranial magnetic stimulation (rTMS) may promote recovery of motor function after stroke by inducing functional reorganization of cortical circuits. The objective of this study was to examine whether multifocal cortical stimulation using a new wearable transcranial rotating permanent magnet stimulator (TRPMS) can promote recovery of motor function after stroke by inducing functional reorganization of cortical circuits. METHODS: Thirty30 patients with chronic ischemic stroke and stable unilateral weakness were enrolled in a Phase 1/2a randomized double-blind sham-controlled clinical trial to evaluate safety and preliminary efficacy. Bilateral hemispheric stimulation was administered for 20 sessions 40 min each over 4 weeks. The primary efficacy endpoint was the change in functional MRI BOLD activation immediately after end of treatment. Secondary efficacy endpoints were clinical scales of motor function, including the Fugl-Meyer motor arm score, ARAT, grip strength, pinch strength, gait velocity, and NIHSS. RESULTS: TRPMS treatment was well-tolerated with no device-related adverse effects. Active treatment produced a significantly greater increase in the number of active voxels on fMRI than sham treatment (median +48.5 vs -30, p = 0.038). The median active voxel number after active treatment was 8.8-fold greater than after sham (227.5 vs 26, p = 0.016). Although the statistical power was inadequate to establish clinical endpoint benefits, numerical improvements were demonstrated in 5 of 6 clinical scales of motor function. The treatment effects persisted over a 3-month duration of follow-up. CONCLUSIONS: Multifocal bilateral TRPMS was safe and showed significant fMRI changes suggestive of functional reorganization of cortical circuits in patients with chronic ischemic stroke. A larger randomized clinical trial is warranted to verify recovery of motor function.

In Situ Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating.

Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The in situ structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how the RNA genome and TEC respond to priming in reoviruses is unknown. Here, we determined the near-atomic-resolution asymmetric structure of ARV in the primed state by cryo-electron microscopy (cryo-EM), revealing the in situ structures of 11 TECs inside each capsid and their interactions with the 11 surrounding double-stranded RNA (dsRNA) genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. The RdRp VP2 and the NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states showed translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel was open in both states, suggesting that channel blocking is not a regulating mechanism between these states in ARV. Instead, the NTPase C-terminal domain appears to regulate RNA translocation between the quiescent and primed states. Taking the data together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining similar mechanisms to coassemble their genome segments, TEC, and capsid proteins into infectious virions.IMPORTANCE Viruses in the family Reoviridae are characterized by the ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genomes with transcriptional enzymes into a multilayered virion and for priming such a virion for transcription are poorly understood. By cryo-EM and novel asymmetric reconstruction, we determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase. The NTPase has a newly discovered domain that translocates the 5' end of plus-sense RNA in segmented dsRNA genomes from the NTPase to polymerase VP2 when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.

Pushing the limits of detection for proteins secreted from single cells using quantum dots.

Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of ∼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.

Building atomic models based on near atomic resolution cryoEM maps with existing tools.

The EMDataBank Validation Challenge was a challenging task for students newly introduced to the cryoEM and molecular modeling fields. However, the competition provided an effective space for student modelers to discover and explore the potentials of atomic modeling and refinement by practicing on published atomic structures. Here, by employing manual molecular modeling programs such as Coot, Phenix, and Chimera, we have regularized and improved three targets. The T20S proteasome and TRPV1 ion channel allowed us to broaden our understanding of these modeling techniques while the 70S ribosome served as a challenge to test the limits of our abilities. We were successful in our efforts to improve each of the models and provide here our cohesive methodology for de novo modeling with and without homology models, which may serve as a starting point for other undergraduates and researchers just entering the realm of cryoEM. Additionally, we provide some constructive criticism to facilitate the introduction of said undergraduates and researchers into cryoEM in the future.

Transition of a Text-Based Insulin Titration Program From a Randomized Controlled Trial Into Real-World Settings: Implementation Study.

BACKGROUND: The Mobile Insulin Titration Intervention (MITI) program helps patients with type 2 diabetes find their correct basal insulin dose without in-person care. Requiring only basic cell phone technology (text messages and phone calls), MITI is highly accessible to patients receiving care in safety-net settings. MITI was shown in a randomized controlled trial (RCT) to be efficacious at a New York City (NYC) safety-net clinic where patients often have challenges coming for in-person care. In 2016, MITI was implemented as usual care at Bellevue Hospital (the site of the original RCT) and at Gouverneur Health (a second NYC safety-net clinic) under 2 different staffing models. OBJECTIVE: This implementation study examined MITI's transition into real-world settings. To understand MITI's flexibility, generalizability, and acceptability among patients and providers, we evaluated whether MITI continued to produce positive outcomes in expanded underserved populations, outside of an RCT setting. METHODS: Patients enrolled in MITI received weekday text messages asking for their fasting blood glucose (FBG) values and a weekly titration call. The goal was for patients to reach their optimal insulin dose (OID), defined either as the dose of once-daily basal insulin required to achieve either an FBG of 80-130 mg/dL (4.4-7.2 mmol/L) or as the reaching of the maximum dose of 50 units. After 12 weeks, if OID was not reached, the patients were asked to return to the clinic for in-person care and titration. MITI program outcomes, clinical outcomes, process outcomes, and patient satisfaction were assessed. RESULTS: MITI was successful at both sites, each with a different staffing model. Providers referred 170 patients to the program-129 of whom (75.9%, 129/170) were eligible. Of these, 113 (87.6%, 113/129) enrolled. Moreover, 84.1% (95/113) of patients reached their OID, and they did so in an average of 24 days. Clinical outcomes show that mean FBG levels fell from 209 mg/dL (11.6 mmol/L) to 141 mg/dL (7.8 mmol/L), P<.001. HbA1c levels fell from 11.4% (101 mmol/mol) to 10.0% (86 mmol/mol), P<.001. Process outcomes show that 90.1% of MITI's text message prompts received a response, nurses connected with patients 81.9% of weeks to provide titration instructions, and 85% of attending physicians made at least one referral to the MITI program. Satisfaction surveys showed that most patients felt comfortable sharing information over text and felt the texts reminded them to take their insulin, check their sugar, and make healthy food choices. CONCLUSIONS: This implementation study showed MITI to have continued success after transitioning from an RCT program into real-world settings. MITI showed itself to be flexible and generalizable as it easily fits into a second site staffed by general medical clinic-registered nurses and remained acceptable to patients and staff who had high levels of engagement with the program.

Probing the solvation structure and dynamics in ionic liquids by time-resolved infrared spectroscopy of 4-(dimethylamino)benzonitrile.

In this work we demonstrate the use of the push-pull model system 4-(dimethylamino)benzonitrile (DMABN) as a convenient molecular probe to investigate the local solvation structure and dynamics by means of time-resolved infrared spectroscopy (TRIR). The photochemical features associated with this system provide several advantages due to the high charge separation between the ground and charge transfer states involving the characteristic nitrile bond, and an excited state lifetime that is long enough to observe the slow solvation dynamics in organic solvents and ionic liquids. The conversion from a locally excited state to an intramolecular charge transfer state (LE-ICT) in ionic liquids shows similar kinetic lifetimes in comparison to organic solvents. This similarity confirms that such conversion depends solely on the intramolecular reorganization of DMABN in the excited state, and not by the dynamics of solvation. In contrast, the relative shift of the ν(CN) vibration during the relaxation of the ICT state reveals two distinct lifetimes that are sensitive to the solvent environment. This study reveals a fast time component which is attributed to the dipolar relaxation of the solvent and a slower time component related to the rotation of the dimethylamino group of DMABN.

Tissue microarray profiling in human heart failure.

Tissue MicroArrays (TMAs) are a versatile tool for high-throughput protein screening, allowing qualitative analysis of a large number of samples on a single slide. We have developed a customizable TMA system that uniquely utilizes cryopreserved human cardiac samples from both heart failure and donor patients to produce formalin-fixed paraffin-embedded sections. Confirmatory upstream or downstream molecular studies can then be performed on the same (biobanked) cryopreserved tissue. In a pilot study, we applied our TMAs to screen for the expression of four-and-a-half LIM-domain 2 (FHL2), a member of the four-and-a-half LIM family. This protein has been implicated in the pathogenesis of heart failure in a variety of animal models. While FHL2 is abundant in the heart, not much is known about its expression in human heart failure. For this purpose, we generated an affinity-purified rabbit polyclonal anti-human FHL2 antibody. Our TMAs allowed high-throughput profiling of FHL2 protein using qualitative and semiquantitative immunohistochemistry that proved complementary to Western blot analysis. We demonstrated a significant relative reduction in FHL2 protein expression across different forms of human heart failure.

Bone Deformities through the Prism of the International Classification of Functioning, Disability and Health in Ambulant Children with Cerebral Palsy: A Systematic Review.

(1) Aim: The aim of this study was to determine the relationship between lower limb bone deformities and body functions, activity, and participation in ambulant children with CP and whether changing bone morphology affects outcomes in these domains. (2) Methods: A systematic literature search (PROSPERO CRD42020208416) of studies reporting correlations between measures of lower limb bone deformities and measures of body function, activity or participation, or post-surgical outcomes in these domains was conducted from 1990 to 2023 in Medline, Scopus, and Cochrane Library. We assessed study quality with the Checklist for Case Series (CCS) and a quality assessment developed by Quebec University Hospital. Meta-analysis was not possible; therefore, descriptive synthesis was performed. (3) Results: A total of 12 of 3373 screened articles were included. No studies evaluated the relationships between bone deformities and activity or participation, or the effect of isolated bone surgery on these domains. Correlations between bone deformities and body functions were poor-to-moderate. Internal hip rotation during gait improved after femoral derotation osteotomy. (4) Conclusions: A shift in paradigm is urgently required for the research and management of bone deformities in children with CP to include the activity and participation domains of the ICF, as well as consider more psychological aspects such as self-image.

Benign Acute Childhood Myositis in a Pediatric Patient Post Influenza B Infection.

Benign acute childhood myositis (BACM) is a mild and self-limited sudden onset of lower extremity pain during or following recovery from a viral illness. It is characterized by difficulty walking due to severe bilateral calf pain, which usually resolves in three days. It is typically appreciated during times of large influenza outbreaks and epidemics. The most severe complication can be rhabdomyolysis without proper treatment and can lead to renal damage and potential renal failure. There are limited reported cases of BACM and therefore no clear guidelines in the treatment or management of the condition. This case is unique in the sense that the patient had leg pain the entire month prior to presentation in the absence of trauma or injury, and it is believed that the pre-existing myalgia may have been exacerbated by an upper respiratory infection (URI) that started a few weeks after the leg pain onset. In addition, this patient's creatine kinase peaked at over 13,000 U/L, which is three to five times higher than the average of other reported children with this condition. The patient is a five-year-old male who presented to the emergency department with bilateral leg pain and difficulty ambulating. His guardian reported that the leg pain began one month prior to presentation and worsened to the point where he could no longer ambulate, following a fever and cough that began one week prior to presentation. A respiratory viral panel was positive for influenza B, and initial creatine kinase (CK) levels were greater than 10,000. A diagnosis of BACM was made, and supportive care was initiated. BACM is an infrequent complication following a viral infection that is typically treatable with hydration management and routine CK monitoring. Symptoms of BACM are usually limited to muscle pain and weakness, but it can progress to rhabdomyolysis and renal failure if not managed properly. It is therefore crucial that physicians monitor CK values daily until a downtrend is noticed and symptoms begin to resolve.

P-element invasion fuels molecular adaptation in laboratory populations of Drosophila melanogaster.

Transposable elements (TEs) are mobile genetic parasites that frequently invade new host genomes through horizontal transfer. Invading TEs often exhibit a burst of transposition, followed by reduced transposition rates as repression evolves in the host. We recreated the horizontal transfer of P-element DNA transposons into a Drosophila melanogaster host and followed the expansion of TE copies and evolution of host repression in replicate laboratory populations reared at different temperatures. We observed that while populations maintained at high temperatures rapidly go extinct after TE invasion, those maintained at lower temperatures persist, allowing for TE spread and the evolution of host repression. We also surprisingly discovered that invaded populations experienced recurrent insertion of P-elements into a specific long non-coding RNA, lncRNA:CR43651, and that these insertion alleles are segregating at unusually high frequency in experimental populations, indicative of positive selection. We propose that, in addition to driving the evolution of repression, transpositional bursts of invading TEs can drive molecular adaptation.

JNK Signalling Regulates Self-Renewal of Proliferative Urine-Derived Renal Progenitor Cells via Inhibition of Ferroptosis.

With a global increase in chronic kidney disease patients, alternatives to dialysis and organ transplantation are needed. Stem cell-based therapies could be one possibility to treat chronic kidney disease. Here, we used multipotent urine-derived renal progenitor cells (UdRPCs) to study nephrogenesis. UdRPCs treated with the JNK inhibitor-AEG3482 displayed decreased proliferation and downregulated transcription of cell cycle-associated genes as well as the kidney progenitor markers-SIX2, SALL1 and VCAM1. In addition, levels of activated SMAD2/3, which is associated with the maintenance of self-renewal in UdRPCs, were decreased. JNK inhibition resulted in less efficient oxidative phosphorylation and more lipid peroxidation via ferroptosis, an iron-dependent non-apoptotic cell death pathway linked to various forms of kidney disease. Our study is the first to describe the importance of JNK signalling as a link between maintenance of self-renewal and protection against ferroptosis in SIX2-positive renal progenitor cells.

Interpreting permeability as a function of free drug fraction: The case studies of cyclodextrins and liposomes.

In order to solubilize poorly soluble active pharmaceutical ingredients, various strategies have been implemented over the years, including the use of nanocarriers, such as cyclodextrins and liposomes. However, improving a drug's apparent solubility does not always translate to enhanced bioavailability. This work aimed to investigate to which extent complexation with cyclodextrins and incorporation into liposomes influence drug in vitro permeability and to find a mechanistic description of the permeation process. For this purpose, we investigated hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and phosphatidylcholine liposomes formulations of three chemically diverse compounds (atenolol, ketoprofen and hydrocortisone). We studied drug diffusion of the formulations by UV-localized spectroscopy and advanced data fitting to extract parameters such as diffusivity and bound-/free drug fractions. We then correlated this information with in vitro drug permeability obtained with the novel PermeaPadⓇ barrier. The results showed that increased concentration of HP-ß-CD leads to increased solubilization of the poorly soluble unionized ketoprofen, as well as hydrocortisone. However, this net increment of apparent solubility was not proportional to the increased flux measured. On the other hand, normalising the flux over the empirical free drug concentration, i.e., the free fraction, gave a meaningful absolute permeability coefficient. The results achieved for the liposomal formulation were consistent with the finding on cyclodextrins. In conclusion, we proved the adequacy and usefulness of our method for calculating free drug fractions in the examined enabling formulations, supporting the validity of the established drug diffusion/permeation theory that the unbounded drug fraction is the main driver for drug permeation across a membrane.

Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells.

Replacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch's membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host's cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.