Development and validation of home-based psychosocial self-management interventions in schizophrenia and related disorders in low-resource settings: A mixed methods approach.

Background: Psychosocial interventions, crucial for recovery in patients with schizophrenia, have often been developed and tested in high income countries. We aimed at developing and validating home-based a booklet based psycho-social intervention with inputs from stakeholders: patients, families, and mental health professionals (MHP) for patients with schizophrenia and related disorders in low resource settings. Methods: We developed a preliminary version of psychosocial intervention booklets based on six themes derived from focus group discussions conducted with patients, families, and MHP. Initially, quantitative assessment of content validity was done by MHP on overall and Content Validity Index of individual items of the specific booklets, followed by in-depth interviews about their views. The booklets were modified based on their inputs. Further, pilot testing of manuals was done on the users - nine pairs of patients and caregivers followed by development of a final version of psycho-social intervention. Results: The percentage content validity of individual modules and overall booklets was ≥78.5% indicating good validity. Most MHP reported that the manuals were relevant and easy to use but were text-heavy, and lengthy. On pilot testing of modified manuals with patients and their family caregivers, majority (77.8%) of them found booklets useful and suggested that there should be separate booklets for both patients and caregivers for providing information and entering separate response for the activities, integrating helpful tips. Language should be simple. Finally, two sets of booklets ("info book" and "workbook") named 'Saksham' (meaning empowered) were created with specific modules (viz., 'Medicine adherence', 'Daily routine', 'Eating right', 'Physical activity', 'Physical health monitoring', 'Self-reliance', and 'Psychoeducation') for patients and caregivers each, in two languages (Hindi and English). Conclusion: Booklets with modules for psychosocial interventions for patients with schizophrenia and their caregivers were developed after establishing content validity and pilot testing.

A frameshift mutation in the murine Prkra gene causes dystonia and exhibits abnormal cerebellar development and reduced eIF2α phosphorylation.

Mutations in Prkra gene, which encodes PACT/RAX cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT/RAX activates protein kinase R (PKR, aka EIF2AK2) by a direct interaction in response to cellular stressors to mediate phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α). Mice homozygous for a naturally arisen, recessively inherited frameshift mutation, Prkra lear-5J exhibit progressive dystonia. In the present study, we investigate the biochemical and developmental consequences of the Prkra lear-5J mutation. Our results indicate that the truncated PACT/RAX protein retains its ability to interact with PKR, however, it inhibits PKR activation. Furthermore, mice homozygous for the mutation have abnormalities in the cerebellar development as well as a severe lack of dendritic arborization of Purkinje neurons. Additionally, reduced eIF2α phosphorylation is noted in the cerebellums and Purkinje neurons of the homozygous Prkra lear-5J mice. These results indicate that PACT/RAX mediated regulation of PKR activity and eIF2α phosphorylation plays a role in cerebellar development and contributes to the dystonia phenotype resulting from this mutation.

Identification of authentic Phyllanthus amarus species in India.

The Phyllanthus is a plant used in the traditional Ayurvedic Medicine system and has more than 800 species. These species grow in the same area and there are chances of adulteration of other species and, incorrect identification may also lead to wrong reports. This study was attempted by Enovate Biolife Pvt. Ltd. to identify authentic Phyllanthus amarus. The nine raw material samples were collected from different populations/suppliers from various zones of India for the study. All the samples were analysed using microscopic and macroscopic ID, and by using the High Performance Thin Layer Chromatography (HPTLC) fingerprint method. The samples collected from the Central zone (Lucknow PA-08, Uttar Pradesh) and the Southern zone (Coimbatore PA-05, and Chennai PA-09, Tamil Nadu) of India were found to be authentic P. amarus by the mentioned identification methods.

Long Noncoding RNA VLDLR-AS1 Levels in Serum Correlate with Combat-Related Chronic Mild Traumatic Brain Injury and Depression Symptoms in US Veterans.

More than 75% of traumatic brain injuries (TBIs) are mild (mTBI) and military service members often experience repeated combat-related mTBI. The chronic comorbidities concomitant with repetitive mTBI (rmTBI) include depression, post-traumatic stress disorder or neurological dysfunction. This study sought to determine a long noncoding RNA (lncRNA) expression signature in serum samples that correlated with rmTBI years after the incidences. Serum samples were obtained from Long-Term Impact of Military-Relevant Brain-Injury Consortium Chronic Effects of Neurotrauma Consortium (LIMBIC CENC) repository, from participants unexposed to TBI or who had rmTBI. Four lncRNAs were identified as consistently present in all samples, as detected via droplet digital PCR and packaged in exosomes enriched for CNS origin. The results, using qPCR, demonstrated that the lncRNA VLDLR-AS1 levels were significantly lower among individuals with rmTBI compared to those with no lifetime TBI. ROC analysis determined an AUC of 0.74 (95% CI: 0.6124 to 0.8741; p = 0.0012). The optimal cutoff for VLDLR-AS1 was ≤153.8 ng. A secondary analysis of clinical data from LIMBIC CENC was conducted to evaluate the psychological symptom burden, and the results show that lncRNAs VLDLR-AS1 and MALAT1 are correlated with symptoms of depression. In conclusion, lncRNA VLDLR-AS1 may serve as a blood biomarker for identifying chronic rmTBI and depression in patients.

Characterization of the bispecific VHH antibody gefurulimab (ALXN1720) targeting complement component 5, and designed for low volume subcutaneous administration.

The bispecific antibody gefurulimab (also known as ALXN1720) was developed to provide patients with a subcutaneous treatment option for chronic disorders involving activation of the terminal complement pathway. Gefurulimab blocks the enzymatic cleavage of complement component 5 (C5) into the biologically active C5a and C5b fragments, which triggers activation of the terminal complement cascade. Heavy-chain variable region antigen-binding fragment (VHH) antibodies targeting C5 and human serum albumin (HSA) were isolated from llama immune-based libraries and humanized. Gefurulimab comprises an N-terminal albumin-binding VHH connected to a C-terminal C5-binding VHH via a flexible linker. The purified bispecific VHH antibody has the expected exact size by mass spectrometry and can be formulated at greater than 100 mg/mL. Gefurulimab binds tightly to human C5 and HSA with dissociation rate constants at pH 7.4 of 54 pM and 0.9 nM, respectively, and cross-reacts with C5 and serum albumin from cynomolgus monkeys. Gefurulimab can associate with C5 and albumin simultaneously, and potently inhibits the terminal complement activity from human serum initiated by any of the three complement pathways in Wieslab assays. Electron microscopy and X-ray crystallography revealed that the isolated C5-binding VHH recognizes the macroglobulin (MG) 4 and MG5 domains of the antigen and thereby is suggested to sterically prevent C5 binding to its activating convertase. Gefurulimab also inhibits complement activity supported by the rare C5 allelic variant featuring an R885H substitution in the MG7 domain. Taken together, these data suggest that gefurulimab may be a promising candidate for the potential treatment of complement-mediated disorders.

Regulation of Human Sortilin Alternative Splicing by Glucagon-like Peptide-1 (GLP1) in Adipocytes.

Type 2 diabetes mellitus is a chronic metabolic disease with no cure. Adipose tissue is a major site of systemic insulin resistance. Sortilin is a central component of the glucose transporter -Glut4 storage vesicles (GSV) which translocate to the plasma membrane to uptake glucose from circulation. Here, using human adipocytes we demonstrate the presence of the alternatively spliced, truncated sortilin variant (Sort_T) whose expression is significantly increased in diabetic adipose tissue. Artificial-intelligence-based modeling, molecular dynamics, intrinsically disordered region analysis, and co-immunoprecipitation demonstrated association of Sort_T with Glut4 and decreased glucose uptake in adipocytes. The results show that glucagon-like peptide-1 (GLP1) hormone decreases Sort_T. We deciphered the molecular mechanism underlying GLP1 regulation of alternative splicing of human sortilin. Using splicing minigenes and RNA-immunoprecipitation assays, the results show that GLP1 regulates Sort_T alternative splicing via the splice factor, TRA2B. We demonstrate that targeted antisense oligonucleotide morpholinos reduces Sort_T levels and improves glucose uptake in diabetic adipocytes. Thus, we demonstrate that GLP1 regulates alternative splicing of sortilin in human diabetic adipocytes.

Luteolin protects DYT-PRKRA cells from apoptosis by suppressing PKR activation.

DYT-PRKRA is a movement disorder caused by mutations in the PRKRA gene, which encodes for PACT, the protein activator of interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR. PACT brings about PKR's catalytic activation by a direct binding in response to stress signals and activated PKR phosphorylates the translation initiation factor eIF2α. Phosphorylation of eIF2α is the central regulatory event that is part of the integrated stress response (ISR), an evolutionarily conserved intracellular signaling network essential for adapting to environmental stresses to maintain healthy cells. A dysregulation of either the level or the duration of eIF2α phosphorylation in response to stress signals causes the normally pro-survival ISR to become pro-apoptotic. Our research has established that the PRKRA mutations reported to cause DYT-PRKRA lead to enhanced PACT-PKR interactions causing a dysregulation of ISR and an increased sensitivity to apoptosis. We have previously identified luteolin, a plant flavonoid, as an inhibitor of the PACT-PKR interaction using high-throughput screening of chemical libraries. Our results presented in this study indicate that luteolin is markedly effective in disrupting the pathological PACT-PKR interactions to protect DYT-PRKRA cells against apoptosis, thus suggesting a therapeutic option for using luteolin to treat DYT-PRKRA and possibly other diseases resulting from enhanced PACT-PKR interactions.

Small molecule targeting long noncoding RNA GAS5 administered intranasally improves neuronal insulin signaling and decreases neuroinflammation in an aged mouse model.

Shifts in normal aging set stage for neurodegeneration and dementia affecting 1 in 10 adults. The study demonstrates that lncRNA GAS5 is decreased in aged and Alzheimer's disease brain. The role and targets of lncRNA GAS5 in the aging brain were elucidated using a GAS5-targeting small molecule NPC86, a frontier in lncRNA-targeting therapeutic. Robust techniques such as molecular dynamics simulation of NPC86 binding to GAS5, in vitro functional assays demonstrating that GAS5 regulates insulin signaling, neuronal survival, phosphorylation of tau, and neuroinflammation via toll-like receptors support the role of GAS5 in maintaining healthy neurons. The study demonstrates the safety and efficacy of intranasal NPC86 treatment in aged mice to improve cellular functions with transcriptomic analysis in response to NPC86. In summary, the study demonstrates that GAS5 contributes to pathways associated with neurodegeneration and NPC86 has tremendous therapeutic potential to prevent the advent of neurodegenerative diseases and dementias.

DYT-PRKRA Mutation P222L Enhances PACT's Stimulatory Activity on Type I Interferon Induction.

DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a PKR activator protein during integrated stress response (ISR) is the best characterized. Previously, we have established that the DYT-PRKRA mutations cause enhanced activation of PKR during ISR to sensitize DYT-PRKRA cells to apoptosis. In this study, we evaluate if the most prevalent substitution mutation reported in DYT-PRKRA patients alters PACT's functional role in induction of type I IFNs via the retinoic acid-inducible gene I (RIG-I) signaling. Our results indicate that the P222L mutation augments PACT's ability to induce IFN ß in response to dsRNA and the basal expression of IFN ß and IFN-stimulated genes (ISGs) is higher in DYT-PRKRA patient cells compared to cells from the unaffected controls. Additionally, IFN ß and ISGs are also induced at higher levels in DYT-PRKRA cells in response to dsRNA. These results offer a new avenue for investigations directed towards understanding the underlying molecular pathomechanisms in DYT-PRKRA.

Understanding needs of stakeholders and outcomes desired from a home-based intervention program for "difficult to treat" schizophrenia and related disorders: A qualitative study.

Background: We aimed to understand the needs of service users - families and patients with schizophrenia and related disorders, and mental health professionals (MHPs) and expectations from a home-based psychosocial intervention program in Indian setting. Materials and Methods: We conducted four focus group discussions (FGDs) with families, patients with schizophrenia and related disorders and MHPs. Two FGDs were conducted with families and one each with the patients and MHPs. Participants in families and MHP group were asked about their primary concerns in caring for the patients, perceived needs of patients and the areas that can be targeted through a home-based psychosocial intervention program. All FGDs were audio-recorded and verbatim transcribed. Content analysis of the data was done to obtain a final list of needs and expected outcomes from a psychosocial intervention supported by families. Results: Six key priority needs were identified for intervention: medication adherence, activities of daily living, promoting physical health, engagement in meaningful work, building of social and support networks and information about all aspects of illness. Priority outcomes identified by MHPs were mostly clinical like symptom reduction, fewer rehospitalisation while families and patients focused more on psychosocial outcomes, such as improvement of wellbeing, having relationships, engagement in meaningful activities, better organization of the day, increased self-respect, reduced stress, lesser interference, and critical comments. All groups suggested that book or mobile app or video could be used. Conclusion: This qualitative study shows that while both clinicians and service users consider recovery from schizophrenia and related disorders to be important, they differ on what they prioritise.

Role of alternatively spliced, pro-survival Protein Kinase C delta VIII (PKCδVIII) in ovarian cancer.

Ovarian cancer is the deadliest malignant disease in women. Protein Kinase C delta (PRKCD; PKCδ) is serine/threonine kinase extensively linked to various cancers. In humans, PKCδ is alternatively spliced to PKCδI and PKCδVIII. However, the specific function of PKCδ splice variants in ovarian cancer has not been elucidated yet. Hence, we evaluated their expression in human ovarian cancer cell lines (OCC): SKOV3 and TOV112D, along with the normal T80 ovarian cells. Our results demonstrate a marked increase in PKCδVIII in OCC compared to normal ovarian cells. Therefore, we elucidated the role of PKCδVIII and the underlying mechanism of its expression in OCC. Using overexpression and knockdown studies, we demonstrate that PKCδVIII increases cellular survival and migration in OCC. Further, overexpression of PKCδVIII in T80 cells resulted in increased expression of Bcl2 and knockdown of PKCδVIII in OCC decreased Bcl2 expression. Using co-immunoprecipitations and immunocytochemistry, we demonstrate nuclear localization of PKCδVIII in OCC and further show increased association of PKCδVIII with Bcl2 and Bcl-xL in OCC. Using PKCδ splicing minigene, mutagenesis, siRNA and antisense oligonucleotides, we demonstrate that increased levels of alternatively spliced PKCδVIII in OCC is regulated by splice factor SRSF2. Finally, we verified that PKCδVIII levels are elevated in samples of human ovarian cancer tissue. The data presented here demonstrate that the alternatively spliced, signaling kinase PKCδVIII is a viable target to develop therapeutics to combat progression of ovarian cancer.

Long Noncoding RNA GAS5 Contained in Exosomes Derived from Human Adipose Stem Cells Promotes Repair and Modulates Inflammation in a Chronic Dermal Wound Healing Model.

Chronic recalcitrant wounds result from delayed or slowed healing processes. Underlying inflammation is a substantial risk factor for impaired dermal wound healing and often leads to chronic wound-related sequelae. Human adipose stem cells (hASCs) have shown tremendous potential in regenerative medicine. The goal of this project was to improve the outcome of chronic wounds by harvesting the exosomes from hASCs for therapeutic intervention. The results demonstrate that long noncoding RNA GAS5 is highly enriched in hASC exosomes and, further, that GAS5 is central to promoting wound repair in vitro. To evaluate the outcome of wound healing in a chronic low-grade inflammatory environment, lipopolysaccharide-treated HDF cells were evaluated for their response to hASC exosome treatment. Ingenuity pathway analysis identified inflammation pathways and genes affected by exosomes in a GAS5-dependent manner. Using siRNA to deplete GAS5 in HDF, the results demonstrated that Toll-like receptor 7 (TLR7) expression levels were regulated by GAS5. Importantly, the results demonstrate that GAS5 regulates inflammatory pathway genes in a chronic inflammation environment. The results presented here demonstrate that hASC exosomes are a viable therapeutic that accelerate the healing of chronic recalcitrant wounds.

Relationship between oxidative stress and lifespan in Daphnia pulex.

Macromolecular damage leading to cell, tissue and ultimately organ dysfunction is a major contributor to aging. Intracellular reactive oxygen species (ROS) resulting from normal metabolism cause most damage to macromolecules and the mitochondria play a central role in this process as they are the principle source of ROS. The relationship between naturally occurring variations in the mitochondrial (MT) genomes leading to correspondingly less or more ROS and macromolecular damage that changes the rate of aging associated organismal decline remains relatively unexplored. MT complex I, a component of the electron transport chain (ETC), is a key source of ROS and the NADH dehydrogenase subunit 5 (ND5) is a highly conserved core protein of the subunits that constitute the backbone of complex I. Using Daphnia as a model organism, we explored if the naturally occurring sequence variations in ND5 correlate with a short or long lifespan. Our results indicate that the short-lived clones have ND5 variants that correlate with reduced complex I activity, increased oxidative damage, and heightened expression of ROS scavenger enzymes. Daphnia offers a unique opportunity to investigate the association between inherited variations in components of complex I and ROS generation which affects the rate of aging and lifespan.

Intranasal delivery of exosomes from human adipose derived stem cells at forty-eight hours post injury reduces motor and cognitive impairments following traumatic brain injury.

The neuroprotective role of human adipose-derived stems cells (hASCs) has raised great interest in regenerative medicine due to their ability to modulate their surrounding environment. Our group has demonstrated that exosomes derived from hASC (hASCexo) are a cell-free regenerative approach to long term recovery following traumatic brain injury (TBI). Previously, we demonstrated the efficacy of exosome treatment with intravenous delivery at 3 h post TBI in rats. Here, we show efficacy of exosomes through intranasal delivery at 48 h post TBI in mice lengthening the therapeutic window of treatment and therefore increasing possible translation to clinical studies. Our findings demonstrate significant recovery of motor impairment assessed by an elevated body swing test in mice treated with exosomes containing MALAT1 compared to both TBI mice without exosomes and exosomes depleted of MALAT1. Significant cognitive improvement was seen in the reversal trial of 8 arm radial arm water maze in mice treated with exosomes containing MALAT1. Furthermore, cortical damage was significantly reduced in mice treated with exosomes containing MALAT1 as well as decreased MHCII+ staining of microglial cells. Mice without exosomes or treated with exosomes depleted of MALAT1 did not show similar recovery. Results demonstrate both inflammation related genes and NRTK3 (TrkC) are target genes modulated by hASC exosomes and further that MALAT1 in hASC exosomes regulates expression of full length TrkC thereby activating the MAPK pathway and promoting recovery. Exosomes are a promising therapeutic approach following TBI with a therapeutic window of at least 48 h and contain long noncoding RNA's, specifically MALAT1 that play a vital role in the mechanism of action.

Social inequalities in accelerated aging among southern U.S. women: an analysis of the biosocial and behavioral pathways linking social determinants to telomere length.

Few studies have examined the biosocial pathways linking socioeconomic status (SES) to accelerated aging in a population-based sample of southern US women. Even fewer have examined the importance of chronic compared to perceived stress in linking SES to women's salivary telomere length (STL). Using data from a probability-based sample of 156 US women and structural equation modeling, we examined three pathways - chronic stress exposure, stress appraisal, and coping behavior - linking SES to STL. SES was positively associated with STL (ßTE = 0.16, p < .05). Everyday discrimination was negatively associated with STL (ßDE = -0.21, p < .05), but perceived stress was positively associated with STL (ßDE = 0.20, p < .05). Current smoking decreased STL (ßDE = -0.19, p < .01). Perceived stress acted to suppress the negative relationship of chronic stress exposure on STL. Given the dearth of STL studies that include measures of both perceived and chronic stress, our study supports the importance of disentangling stress measures and a biosocial approach to the study of accelerated aging.

Regulation of PKR activation and apoptosis during oxidative stress by TRBP phosphorylation.

Transactivation response element RNA-binding protein (TRBP or TARBP2) originally identified as a pro-viral cellular protein in human immunodeficiency virus (HIV) replication is also a regulator of microRNA biogenesis and cellular stress response. TRBP inhibits the catalytic activity of interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) during viral infections and cell stress thereby regulating stress-induced signaling pathways. During cellular stress, PKR is catalytically activated transiently by its protein activator PACT and TRBP inhibits PKR to bring about a timely cellular recovery. We have previously established that TRBP phosphorylated after oxidative stress binds to and inhibits PKR more efficiently promoting cell survival. In this study, we investigated if phosphorylation of TRBP enhances its interaction with PACT to bring about additional PKR inhibition. Our data establishes that phosphorylation of TRBP has no effect on PACT-TRBP interaction and TRBP's inhibitory actions on PKR are mediated exclusively by its enhanced interaction with PKR. Cells lacking TRBP are more sensitive to apoptosis in response to oxidative stress and show persistent PKR activation. These results establish that PKR inhibition by stress-induced TRBP phosphorylation occurs by its direct binding to PKR and is important for preventing apoptosis due to sustained PKR activation.

Identification of Sortilin Alternatively Spliced Variants in Mouse 3T3L1 Adipocytes.

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein-protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort17b. Further, results demonstrate that over-expression of Sort17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome.

Opposite actions of two dsRNA-binding proteins PACT and TRBP on RIG-I mediated signaling.

An integral aspect of innate immunity is the ability to detect foreign molecules of viral origin to initiate antiviral signaling via pattern recognition receptors (PRRs). One such receptor is the RNA helicase retinoic acid inducible gene 1 (RIG-I), which detects and is activated by 5'triphosphate uncapped double stranded RNA (dsRNA) as well as the cytoplasmic viral mimic dsRNA polyI:C. Once activated, RIG-I's CARD domains oligomerize and initiate downstream signaling via mitochondrial antiviral signaling protein (MAVS), ultimately inducing interferon (IFN) production. Another dsRNA binding protein PACT, originally identified as the cellular protein activator of dsRNA-activated protein kinase (PKR), is known to enhance RIG-I signaling in response to polyI:C treatment, in part by stimulating RIG-I's ATPase and helicase activities. TAR-RNA-binding protein (TRBP), which is ∼45% homologous to PACT, inhibits PKR signaling by binding to PKR as well as by sequestration of its' activators, dsRNA and PACT. Despite the extensive homology and similar structure of PACT and TRBP, the role of TRBP has not been explored much in RIG-I signaling. This work focuses on the effect of TRBP on RIG-I signaling and IFN production. Our results indicate that TRBP acts as an inhibitor of RIG-I signaling in a PACT- and PKR-independent manner. Surprisingly, this inhibition is independent of TRBP's post-translational modifications that are important for other signaling functions of TRBP, but TRBP's dsRNA-binding ability is essential. Our work has major implications on viral susceptibility, disease progression, and antiviral immunity as it demonstrates the regulatory interplay between PACT and TRBP IFN production.

The interruption of atypical PKC signaling and Temozolomide combination therapy against glioblastoma.

Current treatment options of glioblastoma include chemotherapy and limited surgical resection. Temozolomide (TMZ) is the current therapeutic choice for chemotherapy. Still, it has severe limitations due to the development of resistance that occurs by genetic modification and constitutive activation of several cell signaling pathways. Therefore, it is essential to develop combination therapy of TMZ with other novel compounds to prevent the development of chemo-resistance. In this study, we used two inhibitors; ICA, an inhibitor of PKC-ι and ζ-Stat, an inhibitor of PKC-ζ. T98G and U87MG glioblastoma cells were treated with either ICA or ζ-stat or TMZ monotherapies, as well as TMZ were combined with either ICA or ζ-stat for five consecutive days. Our in vitro results exhibited that ICA when combined with TMZ, significantly decreased the viability of cancerous cells compared with untreated or TMZ or ICA monotherapies. Additionally, glioblastoma cells were remarkably undergoing apoptosis against the combination treatment of TMZ and ICA nucleotide compared with untreated control cells, as suggested by our Annexin-V/PI flow cytometric analysis. Moreover, the combination of TMZ and ICA also decreased the invasion of glioblastoma cell lines by acting on FAK/Paxillin pathway, as evidenced by scratch assay, transwell invasion assay, Western blot and immunoprecipitation analysis. Furthermore, our in vivo data presented that the combination of ICA and TMZ also reduced glioblastoma tumor growth and volume in mice. These data suggest that atypical PKCs, particularly PKC-ι might be an important therapeutic target as adjuvant therapy in the treatment of glioblastoma.

Multilevel Regulation of Protein Kinase CδI Alternative Splicing by Lithium Chloride.

Lithium chloride (LiCl) is commonly used in treatment of mood disorders; however, its usage leads to weight gain, which promotes metabolic disorders. Protein kinase C delta (PKCδ), a serine/threonine kinase, is alternatively spliced to PKCδI and PKCδII in 3T3-L1 cells. We previously demonstrated that PKCδI is the predominantly expressed isoform in 3T3-L1 preadipocytes. Here, we demonstrate that LiCl treatment decreases PKCδI levels, increases formation of lipid droplets, and increases oxidative stress. Hence, we investigated the molecular mechanisms underlying the regulation of PKCδI alternative splicing by LiCl. We previously demonstrated that the splice factor SFRS10 is essential for PKCδI splicing. Our results demonstrate that glycogen synthase kinase 3 beta (GSK3ß) phosphorylates SFRS10, and SFRS10 is in a complex with long noncoding RNA NEAT1 to promote PKCδI splicing. Using PKCδ splicing minigene and RNA immunoprecipitation assays, our results demonstrate that upon LiCl treatment, NEAT1 levels are reduced, GSK3ß activity is inhibited, and SFRS10 phosphorylation is decreased, which leads to decreased expression of PKCδI. Integration of the GSK3ß signaling pathway with the ribonucleoprotein complex of long noncoding RNA (lncRNA) NEAT1 and SFRS10 enables fine-tuning of PKCδI expression during adipogenesis. Knowledge of the molecular pathways impacted by LiCl provides an understanding of the ascent of obesity as a comorbidity in disease management.