Cdk5 regulates IP3R1-mediated Ca2+ dynamics and Ca2+-mediated cell proliferation.

Loss of cyclin-dependent kinase 5 (Cdk5) in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) increases ER-mitochondria tethering and ER Ca2+ transfer to the mitochondria, subsequently increasing mitochondrial Ca2+ concentration ([Ca2+]mt). This suggests a role for Cdk5 in regulating intracellular Ca2+ dynamics, but how Cdk5 is involved in this process remains to be explored. Using ex vivo primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5-/- mouse embryos, we show here that loss of Cdk5 causes an increase in cytosolic Ca2+concentration ([Ca2+]cyt), which is not due to reduced internal Ca2+ store capacity or increased Ca2+ influx from the extracellular milieu. Instead, by stimulation with ATP that mediates release of Ca2+ from internal stores, we determined that the rise in [Ca2+]cyt in Cdk5-/- MEFs is due to increased inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from internal stores. Cdk5 interacts with the IP3R1 Ca2+ channel and phosphorylates it at Ser421. Such phosphorylation controls IP3R1-mediated Ca2+ release as loss of Cdk5, and thus, loss of IP3R1 Ser421 phosphorylation triggers an increase in IP3R1-mediated Ca2+ release in Cdk5-/- MEFs, resulting in elevated [Ca2+]cyt. Elevated [Ca2+]cyt in these cells further induces the production of reactive oxygen species (ROS), which upregulates the levels of Nrf2 and its targets, Prx1 and Prx2. Cdk5-/- MEFs, which have elevated [Ca2+]cyt, proliferate at a faster rate compared to wt, and Cdk5-/- embryos have increased body weight and size compared to their wt littermates. Taken together, we show that altered IP3R1-mediated Ca2+ dynamics due to Cdk5 loss correspond to accelerated cell proliferation that correlates with increased body weight and size in Cdk5-/- embryos.

Effectiveness of telehealth in preventive care: a study protocol for a randomised controlled trial of tele-exercise programme involving older people with possible sarcopenia or at risk of fall.

BACKGROUND: Continuous loss of muscle mass and strength are the consequences of the ageing process, which increase the risk of falls among older people. Falls can lead to severe consequences such as bone fractures and hampered physical and psychological well-being. Regular exercise is the key to reversing muscle atrophy and relieving sarcopenia. However, the frailty of older people and the recent COVID-19 pandemic may affect their confidence to leave home to attend classes in the community. A feasible and effective alternative should be explored. METHODS: The primary objective is to evaluate the effectiveness of tele-exercise (TE) in relation to physical functioning and exercise adherence among community-dwelling older people at risk of falls in comparison with a community-based group (CB). The secondary objective includes evaluating older people's experience with tele-exercise, emphasizing their psychological welfare, social well-being, and acceptance of the telehealth approach. The design, conduct, and report follow the SPIRIT guidelines (Standard Protocol Items: recommended items to address in a Clinical Trial Protocol and Related Documents). Older people will be recruited from 10 local community centres in Hong Kong and randomly allocated into two groups. All participants will attend the exercise training 3 days per week for 3 months but the mode of delivery will differ, either online as the tele-exercise group (TE) or face-to-face as the community-based group (CB). The outcome measures include muscle strength, physical function, exercise adherence and dropout rate, psychological and social well-being will be assessed at the baseline, and the 3rd, 6th and 12th month. Some participants will be invited to attend focus group interviews to evaluate their overall experience of the tele-exercise training. DISCUSSION: Tele-exercise reduces the barriers to exercise, such as time constraints, inaccessibility to facilities, and the fear of frail older people leaving their homes. Promoting an online home-based exercise programme for older people can encourage them to engage in regular physical activity and increase their exercise adherence even when remaining at home. The use of telehealth can potentially result in savings in cost and time. The final findings will provide insights on delivering exercise via telehealth to older people and propose an exercise delivery and maintenance model for future practice. TRIAL REGISTRATION: Chinese Clinical Trial Registry ( https://www.chictr.org.cn/hvshowprojectEN.html?id=219002&v=1.1 ), registration number: ChiCTR2200063370. Registered on 5 September 2022.

The use of technology for online learning among older adults in Hong Kong.

The use of technology to facilitate better living and learning is gaining popularity worldwide. More and more older adults are technology users and participating in online learning. While there is ample research examining the factors affecting older adults' behavioural intention to use technology more generally, less is known about their views of using technology in online learning environments. Applying the model for the adoption of technology by older adults (MATOA) developed by Kenneth Hsiche Wang et al., the authors of the study presented here investigated the attitudes of older adults towards technology adoption for online learning. They interviewed 20 adults aged 52-73 who had enrolled in a diploma course in a higher educational institution in Hong Kong. The results show that participants held favourable attitudes towards online learning after several practice sessions. While they had confidence in their future use of technology, they stated that the availability of technical and social support affected their intention of continuous learning using technology in online settings.

Utilisation de la technologie par les seniors à Hongkong pour apprendre en ligne ­ Utiliser la technologie pour faciliter l'amélioration de la qualité de vie et l'apprentissage, une activité qui jouit d'un engouement croissant dans le monde entier. De plus en plus de seniors se servent de la technologie pour se livrer à des activités d'apprentissage en ligne. Tandis que de nombreuses recherches portent sur les facteurs qui influent sur l'intention comportementale des seniors à utiliser la technologie d'une manière plus générale, nous savons peu de choses sur leurs points de vue à ce sujet dans les environnements d'apprentissage en ligne. S'appuyant sur le modèle pour l'adoption de la technologie par les seniors (model for the adoption of technology by older adults/MATOA) élaboré par Kenneth Hsiche Wang et coll., les auteurs de la présente étude se sont penchés sur les attitudes des seniors concernant l'adoption de la technologie pour apprendre en ligne. Ils ont interviewé 20 adultes âgés de 52 à 73 ans qui s'étaient inscrits à un cours diplômant dans un établissement d'enseignement supérieur à Hongkong. Les résultats indiquent que les participants se montraient favorables à l'apprentissage en ligne une fois qu'ils avaient participé à plusieurs séances d'entraînement. Tout en se sentant confiants quant à l'utilisation qu'ils feraient à l'avenir de la technologie, ils ont déclaré que la disponibilité d'un soutien technique et social influait sur leur intention d'apprendre en permanence en utilisant la technologie dans un environnement en ligne.

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma-Associated Leptomeningeal Disease.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

Spatial transcriptomics analysis identifies a tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease.

Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. Here, we characterize the tumor microenvironment of LMD and patient-matched extra-cranial metastases using spatial transcriptomics in a small number of clinical specimens (nine tissues from two patients) with extensive in vitro and in vivo validation. The spatial landscape of melanoma LMD is characterized by a lack of immune infiltration and instead exhibits a higher level of stromal involvement. The tumor-stroma interactions at the leptomeninges activate tumor-promoting signaling, mediated through upregulation of SERPINA3. The meningeal stroma is required for melanoma cells to survive in the cerebrospinal fluid (CSF) and promotes MAPK inhibitor resistance. Knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in tumor cell death and re-sensitization to MAPK-targeting therapy. Our data provide a spatial atlas of melanoma LMD, identify the tumor-promoting role of meningeal stroma, and demonstrate a mechanism for overcoming microenvironment-mediated drug resistance in LMD.

Spatial transcriptomics analysis identifies a unique tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease.

Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. The inaccessible nature of the disease site and lack of understanding of the biology of this unique metastatic site are major barriers to developing efficacious therapies for patients with melanoma LMD. Here, we characterize the tumor microenvironment of the leptomeningeal tissues and patient-matched extra-cranial metastatic sites using spatial transcriptomic analyses with in vitro and in vivo validation. We show the spatial landscape of melanoma LMD to be characterized by a lack of immune infiltration and instead exhibit a higher level of stromal involvement. We show that the tumor-stroma interactions at the leptomeninges activate pathways implicated in tumor-promoting signaling, mediated through upregulation of SERPINA3 at the tumor-stroma interface. Our functional experiments establish that the meningeal stroma is required for melanoma cells to survive in the CSF environment and that these interactions lead to a lack of MAPK inhibitor sensitivity in the tumor. We show that knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in re-sensitization of the tumor to MAPK-targeting therapy and tumor cell death in the leptomeningeal environment. Our data provides a spatial atlas of melanoma LMD, identifies the tumor-promoting role of meningeal stroma, and demonstrates a mechanism for overcoming microenvironment-mediated drug resistance unique to this metastatic site.

Hepatic haematoma after shockwave lithotripsy for renal stones.

Extracorporeal shock wave lithotripsy (SWL) is a non-invasive procedure for urolithiasis. Only a very small portion of patients suffer from post-SWL haematoma and most of them have perinephric haematoma formation. We present two patients who developed subcapsular hepatic haematomas after SWL, followed by a review of the literature on the condition.

A preclinical model of patient-derived cerebrospinal fluid circulating tumor cells for experimental therapeutics in leptomeningeal disease from melanoma.

BACKGROUND: Leptomeningeal disease (LMD) occurs as a late complication of several human cancers and has no rationally designed treatment options. A major barrier to developing effective therapies for LMD is the lack of cell-based or preclinical models that recapitulate human disease. Here, we describe the development of in vitro and in vivo cultures of patient-derived cerebrospinal fluid circulating tumor cells (PD-CSF-CTCs) from patients with melanoma as a preclinical model to identify exploitable vulnerabilities in melanoma LMD. METHODS: CSF-CTCs were collected from melanoma patients with melanoma-derived LMD and cultured ex vivo using human meningeal cell-conditioned media. Using immunoassays and RNA-sequencing analyses of PD-CSF-CTCs, molecular signaling pathways were examined and new therapeutic targets were tested for efficacy in PD-CSF-CTCs preclinical models. RESULTS: PD-CSF-CTCs were successfully established both in vitro and in vivo. Global RNA analyses of PD-CSF-CTCs revealed several therapeutically tractable targets. These studies complimented our prior proteomic studies highlighting IGF1 signaling as a potential target in LMD. As a proof of concept, combining treatment of ceritinib and trametinib in vitro and in vivo demonstrated synergistic antitumor activity in PD-CSF-CTCs and BRAF inhibitor-resistant melanoma cells. CONCLUSIONS: This study demonstrates that CSF-CTCs can be grown in vitro and in vivo from some melanoma patients with LMD and used as preclinical models. These models retained melanoma expression patterns and had signaling pathways that are therapeutically targetable. These novel models/reagents may be useful in developing rationally designed treatments for LMD.

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the "Murine Ommaya." As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Single-Cell Characterization of the Immune Microenvironment of Melanoma Brain and Leptomeningeal Metastases.

PURPOSE: Melanoma brain metastases (MBM) and leptomeningeal melanoma metastases (LMM) are two different manifestations of melanoma CNS metastasis. Here, we used single-cell RNA sequencing (scRNA-seq) to define the immune landscape of MBM, LMM, and melanoma skin metastases. EXPERIMENTAL DESIGN: scRNA-seq was undertaken on 43 patient specimens, including 8 skin metastases, 14 MBM, and 19 serial LMM specimens. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by IHC and flow cytometry. Association analyses were undertaken to identify immune cell subsets correlated with overall survival. RESULTS: The LMM microenvironment was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases. An LMM patient with long-term survival demonstrated an immune repertoire distinct from that of poor survivors and more similar to normal cerebrospinal fluid (CSF). Upon response to PD-1 therapy, this extreme responder showed increased levels of T cells and dendritic cells in their CSF, whereas poor survivors showed little improvement in their T-cell responses. In MBM patients, therapy led to increased immune infiltrate, with similar T-cell transcriptional diversity noted between skin metastases and MBM. A correlation analysis across the entire immune landscape identified the presence of a rare population of dendritic cells (DC3) that was associated with increased overall survival and positively regulated the immune environment through modulation of activated T cells and MHC expression. CONCLUSIONS: Our study provides the first atlas of two distinct sites of melanoma CNS metastases and defines the immune cell landscape that underlies the biology of this devastating disease.

Relationships between Physical and Social Behavioural Changes and the Mental Status of Homebound Residents in Hong Kong during the COVID-19 Pandemic.

In Hong Kong, social distancing has been adopted in order to minimise the spread of COVID-19. This study aims to examine the changes in physical health, mental health, and social well-being experienced by local residents who were homebound during the pandemic. An online questionnaire in both Chinese and English versions was completed by 590 eligible participants from 24 April to 13 May 2020. The questionnaire found that individuals aged 18 to 25 years spent more time resting and relaxing but experienced more physical strain. Working status was associated with social contact, with participants working full-time jobs scoring higher in "maintaining social communication via electronic means" and "avoiding social activities outside the home". Additionally, approximately one third of the participants (29.7%) had moderate to severe depression, and participants aged 18 to 25 were found to have higher scores in PHQ-9. Changes in physical health and social contact were significantly associated with developing depressive symptoms. From the results, it is clear that the COVID-19 pandemic has the potential to exert a negative impact on the mental health status of individuals.

Proteomic Analysis of CSF from Patients with Leptomeningeal Melanoma Metastases Identifies Signatures Associated with Disease Progression and Therapeutic Resistance.

PURPOSE: The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the cerebrospinal fluid (CSF) from patients with LMM to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells. EXPERIMENTAL DESIGN: A total of 45 serial CSF samples were collected from 16 patients, 8 of these with confirmed LMM. Of those with LMM, 7 had poor survival (<4 months) and one was an extraordinary responder (still alive with survival >35 months). CSF samples were analyzed by mass spectrometry and incubated with melanoma cells that were subjected to RNA sequencing (RNA-seq) analysis. Functional assays were performed to validate the pathways identified. RESULTS: Mass spectrometry analyses showed the CSF of most patients with LMM to be enriched for pathways involved in innate immunity, protease-mediated damage, and IGF-related signaling. All of these were anticorrelated in the extraordinary responder. RNA-seq analysis showed CSF to induce PI3K/AKT, integrin, B-cell activation, S-phase entry, TNFR2, TGFß, and oxidative stress responses in the melanoma cells. ELISA assays confirmed that TGFß expression increased in the CSF of patients progressing with LMM. CSF from poorly responding patients conferred tolerance to BRAF inhibitor therapy in apoptosis assays. CONCLUSIONS: These analyses identified proteomic/transcriptional signatures in the CSF of patients who succumbed to LMM. We further showed that the CSF from patients with LMM has the potential to modulate BRAF inhibitor responses and may contribute to drug resistance.See related commentary by Glitza Oliva and Tawbi, p. 2083.

Enhancement of Peripheral Nerve Regrowth by the Purine Nucleoside Analog and Cell Cycle Inhibitor, Roscovitine.

Peripheral nerve regeneration is a slow process that can be associated with limited outcomes and thus a search for novel and effective therapy for peripheral nerve injury and disease is crucial. Here, we found that roscovitine, a synthetic purine nucleoside analog, enhances neurite outgrowth in neuronal-like PC12 cells. Furthermore, ex vivo analysis of pre-injured adult rat dorsal root ganglion (DRG) neurons showed that roscovitine enhances neurite regrowth in these cells. Likewise, in vivo transected sciatic nerves in rats locally perfused with roscovitine had augmented repopulation of new myelinated axons beyond the transection zone. By mass spectrometry, we found that roscovitine interacts with tubulin and actin. It interacts directly with tubulin and causes a dose-dependent induction of tubulin polymerization as well as enhances Guanosine-5'-triphosphate (GTP)-dependent tubulin polymerization. Conversely, roscovitine interacts indirectly with actin and counteracts the inhibitory effect of cyclin-dependent kinases 5 (Cdk5) on Actin-Related Proteins 2/3 (Arp2/3)-dependent actin polymerization, and thus, causes actin polymerization. Moreover, in the presence of neurotrophic factors such as nerve growth factor (NGF), roscovitine-enhanced neurite outgrowth is mediated by increased activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways. Since microtubule and F-actin dynamics are critical for axonal regrowth, the ability of roscovitine to activate the ERK1/2 and p38 MAPK pathways and support polymerization of tubulin and actin indicate a major role for this purine nucleoside analog in the promotion of axonal regeneration. Together, our findings demonstrate a therapeutic potential for the purine nucleoside analog, roscovitine, in peripheral nerve injury.

Treatment-Resistant Schizophrenia Patients Show Elevated Anterior Cingulate Cortex Glutamate Compared to Treatment-Responsive.

INTRODUCTION: Resistance to antipsychotic treatment is a significant clinical problem in patients with schizophrenia with approximately 1 in 3 showing limited or no response to repeated treatments with antipsychotic medication. The neurobiological basis for treatment resistance is unknown but recent evidence implicates glutamatergic function in the anterior cingulate cortex. We examined glutamate levels of chronically ill treatment-resistant patients directly compared to treatment-responsive patients. METHODS: We acquired proton magnetic resonance spectroscopy (1H-MRS) at 3 Tesla from 21 treatment-resistant and 20 treatment-responsive patients. All participants had a DSM-IV diagnosis of schizophrenia. Treatment-resistant patients were classified using the modified Kane criteria. The groups were matched for age, sex, smoking status, and illness duration. RESULTS: Glutamate to creatine ratio levels were higher in treatment-resistant patients (Mean [SD] = 1.57 [0.24]) than in treatment-responsive patients (Mean[SD] = 1.38 [0.23]), (T[35] = 2.34, P = .025, 2-tailed), with a large effect size of d = 0.76. A model assuming 2 populations showed a 25% improvement in the fit of the Akaike weights (0.55) over a model assuming 1 population (0.44), producing group values almost identical to actual group means. DISCUSSION: Increased anterior cingulate glutamate level is associated with treatment-resistant schizophrenia. This appears to be a stable neurobiological trait of treatment-resistant patients. We discuss possible explanations for glutamatergic dysfunction playing a significant role in resistance to conventional antipsychotic treatments, which are all dopamine-2 receptor blockers. Our findings suggest that glutamatergic treatments may be particularly effective in resistant patients and that 1H-MRS glutamate indices can potentially have clinical use.

Lasting personality pathology following exposure to catastrophic trauma in adults: systematic review.

BACKGROUND: By definition, personality disorders (PDs) are evident in late childhood and adolescence, but evidence for personality pathology occurring after adolescence is unclear. AIM: We aimed to review extant literature on personality change following exposure to catastrophic trauma in adults in order to identify the prevalence and clinical features of any long-term personality pathology. METHOD: Relevant studies were identified by searching three bibliographic databases (MEDLINE, EMBASE and PsychINFO) from inception to November 2011 using terms related to personality and trauma. RESULTS: No prospective studies that investigated long-term personality change following exposure to trauma in adults were found. Two retrospective studies reported the prevalence of enduring personality change of 2.6% and 6% (weighted prevalence 4.6%, 95% confidence interval 3.4-6.3%), and one study reported 20% increase in adult-onset antisocial behaviour following exposure to trauma. Findings from cross-sectional studies that examined the prevalence of PDs in people exposed to catastrophic trauma reported that Cluster C and Cluster A were the most common with avoidant, paranoid and obsessive-compulsive PDs among those most frequently reported. CONCLUSION: A minority of adults who are exposed to severe trauma appear to go on to develop significant personality pathology. The observed personality disturbance is multifarious and more extensive than the prototype described in ICD-10.