Crocus sativus L. Causes a Non Apoptotic Calpain Dependent Death in C6 Rat Glioma Cells, Exhibiting a Synergistic Effect with Temozolomide.

Crocus sativus L., a dietary herb, has been used for various diseases including cancer. This is an in vitro study investigating the antineoplastic effect of the extract of the plant against C6 glioma rat cell line. The mechanism of cellular death and the synergistic effect of the extract with the alkylating agent temozolomide (TMZ) were investigated. Cellular viability was examined in various concentrations of the extract alone or in combination with TMZ. Apoptosis was determined with flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and autophagy by western blotting of the light chain 3 (LC3)-II. Cellular viability was reduced after exposure to the extract with half maximal inhibition concentration at 3 mg/ml. Flow cytometry and TUNEL assay suggested that the extract does not induce apoptosis. Moreover, their combination increased the ratio dead/apoptotic cells 10-fold (P < 0.001). LC3-II protein levels reduced after Crocus extract while this effect was reversed when the calpain inhibitor MDL28170 was added, suggesting a calpain-dependent death possibly through autophagy. We concluded that the extract of Crocus increases dead cell number after 48 h of exposure. Our results suggest that the cell undergoes calpain-dependent programmed cell death while co-exposure to Crocus extract and TMZ enhances the antineoplastic effect of the latter.

Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells.

BACKGROUND/AIMS: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. METHODS: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. RESULTS: Administration of glutamate in PC12 cells in doses as low as 10 µM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. CONCLUSION: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

Weight gain, gender, and antipsychotics: a disproportionality analysis of the FDA Adverse Event Reporting System database (FAERS).

INTRODUCTION: Side effects are a very important aspect of antipsychotic treatments. Weight gain is an important side effect that jeopardizes the uninterrupted therapy administration, especially in patients with psychiatric conditions. This case-non-case pharmacovigilance study aims at investigating in a real-world adverse event reporting system whether several antipsychotics increase the risk of weight gain reporting, and the differences among men and women as far as weight gain as a reported adverse event is concerned. AREAS COVERED: Adverse event reports submitted to the FDA Adverse Event Reporting System of the Food and Drug Administration of the United States (FAERS) of 24 major antipsychotics were extracted, cleaned, and analyzed to determine which of these drugs were correlated with weight gain. The Reported Odds Ratio (ROR) and the adjusted Reported Odds Ratio (aROR) were calculated for each antipsychotic using logistic regression models. Demographics like age, gender, and concomitant insulin use were taken into consideration for each drug. EXPERT OPINION: Women had a statistically significant increase in weight gain reporting compared to men, while the men's group was associated with a reduced weight gain reporting in every antipsychotics in the logistic regression analyses. Out of the 24 antipsychotics included in our analysis, Aripiprazole, Brexpiprazole, Olanzapine, and Haloperidol had statistically significantly more weight increase reporting compared to the others.

Lung Cancer Proteogenomics: Shaping the Future of Clinical Investigation.

BACKGROUND: Lung cancer is associated with a high incidence of mortality worldwide. Molecular mechanisms governing the disease have been explored by genomic studies; however, several aspects remain elusive. The integration of genomic profiling with in-depth proteomic profiling has introduced a new dimension to lung cancer research, termed proteogenomics. The aim of this review article was to investigate proteogenomic approaches in lung cancer, focusing on how elucidation of proteogenomic features can evoke tangible clinical outcomes. METHODS: A strict methodological approach was adopted for study selection and key article features included molecular attributes, tumor biomarkers, and major hallmarks involved in oncogenesis. RESULTS: As a consensus, in all studies it becomes evident that proteogenomics is anticipated to fill significant knowledge gaps and assist in the discovery of novel treatment options. Genomic profiling unravels patient driver mutations, and exploration of downstream effects uncovers great variability in transcript and protein correlation. Also, emphasis is placed on defining proteogenomic traits of tumors of major histological classes, generating a diverse portrait of predictive markers and druggable targets. CONCLUSIONS: An up-to-date synthesis of landmark lung cancer proteogenomic studies is herein provided, underpinning the importance of proteogenomics in the landscape of personalized medicine for combating lung cancer.

COVID-19 vaccines adverse events: potential molecular mechanisms.

COVID-19 is an infectious disease caused by a single-stranded RNA (ssRNA) virus, known as SARS-CoV-2. The disease, since its first outbreak in Wuhan, China, in December 2019, has led to a global pandemic. The pharmaceutical industry has developed several vaccines, of different vector technologies, against the virus. Of note, among these vaccines, seven have been fully approved by WHO. However, despite the benefits of COVID-19 vaccination, some rare adverse effects have been reported and have been associated with the use of the vaccines developed against SARS-CoV-2, especially those based on mRNA and non-replicating viral vector technology. Rare adverse events reported include allergic and anaphylactic reactions, thrombosis and thrombocytopenia, myocarditis, Bell's palsy, transient myelitis, Guillen-Barre syndrome, recurrences of herpes-zoster, autoimmunity flares, epilepsy, and tachycardia. In this review, we discuss the potential molecular mechanisms leading to these rare adverse events of interest and we also attempt an association with the various vaccine components and platforms. A better understanding of the underlying mechanisms, according to which the vaccines cause side effects, in conjunction with the identification of the vaccine components and/or platforms that are responsible for these reactions, in terms of pharmacovigilance, could probably enable the improvement of future vaccines against COVID-19 and/or even other pathological conditions.

Biopharmaceuticals against substance use disorders - Present and future.

BACKGROUND AND OBJECTIVES: Pharmacological treatments available for substance use disorder (SUD) focus on pharmacodynamics, agonizing or antagonizing the drug of abuse (DOA) on receptor level. Drawbacks of this approach include the reliance on long-term patient compliance, on-target off-site effects, perpetuation of addiction and unavailability for many DOAs. Newer, pharmacokinetic approaches are needed that restrict DOA's access to the brain or disrupt DOA-instated brain changes maintaining addiction. Biotechnology might be able to provide the right biopharmaceutical tools to deliver a fine-tuned solution with less side effects compared to currently available treatments. METHODS: This review examines the available literature on biopharmaceuticals developed to treat SUD. RESULTS: Active and passive immunization, metabolic enhancers that augment DOA metabolism and clearance, as well as genetic/epigenetic modulation are promising next generation SUD treatments. Active immunization relies on production of antidrug antibodies by means of vaccination, while passive immunization constitutes of exogenous administration of such antibodies. Metabolic enhancers include drug-specific metabolizing enzymes that can be administered or secreted by modified skin grafts, as well as catalytic antibodies that hasten DOA metabolism. Nanotechnological advances can also allow for brain delivery of siRNAs, mRNAs or DNA in order to modulate central, common in all addictions, genetic or epigenetic targets attenuating drug seeking behavior and reversing drug-induced brain changes. CONCLUSIONS: and Scientific Significance: Biopharmaceuticals can in the future complement or even replace traditional pharmacodynamics approaches in SUD treatment. While passive and active immunization biopharmaceuticals have entered human clinical trials, metabolic enhancers and genetic approaches are at the preclinical level.

mRNA in the Context of Protein Replacement Therapy.

Protein replacement therapy is an umbrella term used for medical treatments that aim to substitute or replenish specific protein deficiencies that result either from the protein being absent or non-functional due to mutations in affected patients. Traditionally, such an approach requires a well characterized but arduous and expensive protein production procedure that employs in vitro expression and translation of the pharmaceutical protein in host cells, followed by extensive purification steps. In the wake of the SARS-CoV-2 pandemic, mRNA-based pharmaceuticals were recruited to achieve rapid in vivo production of antigens, proving that the in vivo translation of exogenously administered mRNA is nowadays a viable therapeutic option. In addition, the urgency of the situation and worldwide demand for mRNA-based medicine has led to an evolution in relevant technologies, such as in vitro transcription and nanolipid carriers. In this review, we present preclinical and clinical applications of mRNA as a tool for protein replacement therapy, alongside with information pertaining to the manufacture of modified mRNA through in vitro transcription, carriers employed for its intracellular delivery and critical quality attributes pertaining to the finished product.

Ceramides during Pregnancy and Obstetrical Adverse Outcomes.

Ceramides are a group of sphingolipids located in the external plasma membrane layer and act as messengers in cellular pathways such as inflammatory processes and apoptosis. Plasma ceramides are biomarkers of cardiovascular disease, type 2 diabetes mellitus, Alzheimer's disease, various autoimmune conditions and cancer. During pregnancy, ceramides play an important role as stress mediators, especially during implantation, delivery and lactation. Based on the current literature, plasma ceramides could be potential biomarkers of obstetrical adverse outcomes, although their role in metabolic pathways under such conditions remains unclear. This review aims to present current studies that examine the role of ceramides during pregnancy and obstetrical adverse outcomes, such as pre-eclampsia, gestational diabetes mellitus and other complications.

Effects of GABAergic Agents on Multiple Sclerosis. A Narrative Review of In-vivo Models.

BACKGROUND: Multiple sclerosis (MS) is a lifelong deteriorating disease characterized by multiple heterogeneous symptoms. Being an autoimmune disease of the central nervous system, mainly affecting the myelin sheath of the nerves ordinarily results in neurological symptoms. GABA has numerous effects on the immune cells, altering cytokine production, cell migration and proliferation. Immune cells express GABA receptors making GABA an inflammation modulator. Therefore, GABAergic- associated agents could provide a compatible add-on therapy for MS patients alleviating their symptoms and providing better quality years. OBJECTIVE: This review aims to highlight and provide evidence of the potential benefits of a secondary treatment option in MS patients, aiming to better manage this disease. METHODS: We conducted a literature search through PubMed, Scopus and Google Scholar for GABA agonists, antagonists and modulators used in the in vivo model of experimental autoimmune encephalomyelitis (EAE), taking into consideration certain inclusion and exclusion criteria. RESULTS: In vivo studies for GABA-a and GABA-b agonists and modulators showed regulation of the autoimmune response in EAE mice. Increased preservation of myelinated sensitive fibers and diminished axonal damage in the CNS was also demonstrated. Further, decreased mononuclear inflammatory infiltration, pro-inflammatory cytokines reduction and reduced levels of Reactive oxygen species (ROS) were also reported. Biological results included decreased peak disease severity, duration, clinical scores and EAE incidence in the treatment groups. CONCLUSION: GABA agonists and modulators efficiently challenged different aspects of disease pathophysiology in vivo models of EAE. The studies showed a significant relevance of neuroprotection via modulation of the autoimmune response in EAE rats, indicating that they should be considered proper therapeutic candidates for clinical use, while also further clinical studies could empower their administration in clinical practice.

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles.

In the quest for a formidable weapon against the SARS-CoV-2 pandemic, mRNA therapeutics have stolen the spotlight. mRNA vaccines are a prime example of the benefits of mRNA approaches towards a broad array of clinical entities and druggable targets. Amongst these benefits is the rapid cycle "from design to production" of an mRNA product compared to their peptide counterparts, the mutability of the production line should another target be chosen, the side-stepping of safety issues posed by DNA therapeutics being permanently integrated into the transfected cell's genome and the controlled precision over the translated peptides. Furthermore, mRNA applications are versatile: apart from vaccines it can be used as a replacement therapy, even to create chimeric antigen receptor T-cells or reprogram somatic cells. Still, the sudden global demand for mRNA has highlighted the shortcomings in its industrial production as well as its formulation, efficacy and applicability. Continuous, smart mRNA manufacturing 4.0 technologies have been recently proposed to address such challenges. In this work, we examine the lab and upscaled production of mRNA therapeutics, the mRNA modifications proposed that increase its efficacy and lower its immunogenicity, the vectors available for delivery and the stability considerations concerning long-term storage.

Influence of conception and delivery mode on stress response marker Oct4B1 and imprinted gene expression related to embryo development: A cohort study.

BACKGROUND: Recent scientific data support that the mode of conception and delivery may influence epigenetic regulation and therefore embryo development. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel variant of OCT4 with yet unknown biological function, is suggested to have a potential role in mediating cellular stress response. Furthermore, Insulinlike Growth Factor 2 (IGF2), Mesoderm-specific Transcript (MEST) and paternally expressed gene 10 (PEG10) are genes known as imprinted and are regulated via means of epigenetic regulation. The influence of delivery mode and conception on epigenetic regulation is an active research field. OBJECTIVE: Our aim was to correlate the expression level of Oct4B1 and the expression and methylation level of IGF2, MEST, and PEG10 imprinted genes with the mode of delivery and conception in the umbilical cord blood of newborns. MATERIALS AND METHODS: Samples of umbilical cord blood from infants born after vaginal delivery, caesarean section (CS) with the infant in cephalic position and CS due to breech position were examined. Furthermore, the investigation included infants conceived through means of assisted reproductive technology. RESULTS: No statistically significant differences were found in mRNA expression levels between different modes of conception and delivery (p = 0.96). Oct4B1, IGF2, MEST, and PEG10 expression levels do not seem to be significantly affected by different modes of conception and delivery. CONCLUSION: These results indicate that the expression and methylation patterns of Oct4B1, IGF2, MEST and PEG10 in umbilical cord blood are not affected by the conception and delivery mode.

Traumatic Elbow Mediolateral Investigatory Screening: The value of the sitting axial mediolateral projection in the diagnosis of radial head fractures.

BACKGROUND: Elbow injuries are frequently encountered in hospital practice, while radial head fractures are the commonest result of such trauma. Diagnosis of radial head fractures is based on clinical and radiographic evaluation with a number of X-ray projections being available. A combination of views is chosen, comprising of routine elbow series screenings and modified techniques, as well as the assistance of the fat-pat sign; never the less fractures can still be occult on X-rays. PURPOSE: This article aims to demonstrate the diagnostic value of the sitting axial mediolateral projection, which in our hospital is referred to as the Traumatic Elbow Mediolateral Investigatory Screening (TEMIS). This projection has been successfully used in our hospital repeatedly for the identification of occult fractures that were missed by other projections. MATERIALS AND METHODS: 62 patients who presented to our Emergency Department due to trauma in the elbow over a six-month period were X-rayed for the identification of fractures. Projections included an anteroposterior, a lateral and TEMIS. RESULTS: Radial head fractures (Mason type I) were identified in 14 out of 62 patients. 2 of these were only visible on the TEMIS projection, while they were missed by the other two views. CONCLUSION: Trauma to the elbow is a common type of injury. Minor fractures can be expected to heal with good results; complications, however, should not be underestimated. Improper healing can result in a permanently malfunctioning joint with restricted movement and stiffness. To avoid such consequences the choice of a reliable screening projection is significant. In our case the diagnostic value of the TEMIS projection lies in the fact that it shows all fractures seen on routine screenings and reveals fractures missed by them, also being well tolerated by patients due to comfortable arm placement during the screening. All in all, when a fracture is suspected on the basis of clinical signs but no radiographic evidence is seen on conventional screenings, the TEMIS view is recommended for further investigation.

Closed Rupture of Anterior Tibialis Tendon after Minor Injury without Medical History: A Surgical Repair.

INTRODUCTION: Closed rupture of the anterior tibial tendon is uncommon, with only a few cases being documented in literature. It usually happens in the forceful plantar flexion of the foot while in eversion and the simultaneous contraction of the anterior tibialis muscle. CARE REPORT: We present the case of a 65-year-old man with closed rupture of the tibialis anterior tendon after injury who was treated by primary surgical repair. The surgical technique as well as the rehabilitation protocol are described in detail. CONCLUSION: Primary surgical reconstruction repair is indicated in younger, active individuals. It appears that early diagnosis and treatment provides the best clinical results, while delayed treatment usually requires the use of a tendon graft in combination with tendon transfers, giving inferior results compared to primary tendon repair.

Assessment of the implementation of the Baby-Friendly Hospital Initiative in Greece.

INTRODUCTION: This report aims to outline the views of health professionals, working in maternity clinics, on the usefulness, benefits and difficulties of integrating the Baby-Friendly Hospital Initiative (BFHI), and to illustrate their degree of awareness about it and extent to which they are informed about BHFI. METHODS: The method of qualitative research was used to conduct the study using an open-ended questionnaire and interview technique. Sixteen health professionals working in 4 hospitals and maternity clinics in Thessaloniki (Ippokrateio, Papageorgiou, Diabalkaniko, and Genesis) participated in the interviews with equal participation of individuals in the sample. RESULTS: The extent to which respondents are informed about the Βaby-Friendly Hospital Initiative is relatively satisfactory, but there is no comprehensive knowledge of the steps required by the Harmonisation Directive. Harmonisation with the guidelines proposed by the BFHI is expected to improve the quality of obstetric clinic services and lead to a reduction in costs. However, there are particular difficulties in adapting hospitals to baby-friendly practices, particularly concerning the resources required. CONCLUSIONS: Hospitals need to make significant changes in organisation, resource efficiency and utilization, and staff training, to fully integrate the BFHI, as integration of procedures is very limited and no research has been conducted in this area in Greece.

Hyaline cartilage next generation implants from adipose-tissue-derived mesenchymal stem cells: Comparative study on 3D-printed polycaprolactone scaffold patterns.

We used additive manufacturing to fabricate 3D-printed polycaprolactone scaffolds of different geometry topologies and porosities. We present a comparative analysis of hyaline cartilage development from adipose-tissue-derived mesenchymal stem cells (ADMSCs) on three different, newly designed scaffold geometry patterns. The first scaffold design (MESO) was based on a rectilinear layer pattern. For the second pattern (RO45), we employed a 45° rotational layer loop. The design for the third scaffold (3DHC) was a three-dimensional honeycomb-like pattern with a hexagonal cellular distribution and small square shapes. We examined cell proliferation, colonization, and differentiation, in relation to the scaffold's structure, as well as to the mechanical properties of the final constructs. We gave emphasis on the scaffolds, both microarchitecture and macroarchitecture, for optimal and enhanced chondrogenic differentiation, as an important parameter, not well studied in the literature. Among the three patterns tested, RO45 was the most favourable for chondrogenic differentiation, whereas 3DHC better supported cell proliferation and scaffold penetration, exhibiting also the highest rate of increase onto the mechanical properties of the final construct. We conclude that by choosing the optimal scaffold architecture, the resulting properties of our cartilaginous constructs can better approximate those of the physiological cartilage.

Comorbid presentation of syringomyelia and Guillain-Barre syndrome, attributed to mycoplasma, in a 6-year-old female patient.

Syringomyelia is the development of a fluid-filled cavity or syrinx within the spinal cord that can cause loss of sensation and muscle spasticity. Guillain-Barre syndrome (GBS) is a postinfection autoimmune disease, classified as an acute polyneuropathy. This report describes the emergency admission of a 6-year-old girl presenting with sudden pallor and pain in both lower limbs. The patient's reflexes were normal, as were the results of her sonography, radiography and biochemical tests; however, spinal MRI revealed extensive compartmentalised syringomyelia extending from C2 to T3. A sensory and motor nerve conduction study revealed a demyelinating type motor polyneuropathy which, along with positive Mycoplasma pneumoniae test, was suggestive of GBS. Intravenous immunoglobulin infusion showed excellent results. In conclusion, we report a rare paediatric case of syringomyelia coexisting with GBS. It is important to bear in mind the possibility of other coexisting diseases even if MRI reveals definitive characteristics of another condition.

Researching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study.

Although glutamate is one of the most important excitatory neurotransmitters of the central nervous system, its excessive extracellular concentration leads to uncontrolled continuous depolarization of neurons, a toxic process called, excitotoxicity. In excitotoxicity glutamate triggers the rise of intracellular Ca(2+) levels, followed by up regulation of nNOS, dysfunction of mitochondria, ROS production, ER stress, and release of lysosomal enzymes. Excessive calcium concentration is the key mediator of glutamate toxicity through over activation of ionotropic and metabotropic receptors. In addition, glutamate accumulation can also inhibit cystine (CySS) uptake by reversing the action of the CySS/glutamate antiporter. Reversal of the antiporter action reinforces the aforementioned events by depleting neurons of cysteine and eventually glutathione's reducing potential. Various cell lines have been employed in the pursuit to understand the mechanism(s) by which excitotoxicity affects the cells leading them ultimately to their demise. In some cell lines glutamate toxicity is exerted mainly through over activation of NMDA, AMPA, or kainate receptors whereas in other cell lines lacking such receptors, the toxicity is due to glutamate induced oxidative stress. However, in the greatest majority of the cell lines ionotropic glutamate receptors are present, co-existing to CySS/glutamate antiporters and metabotropic glutamate receptors, supporting the assumption that excitotoxicity effect in these cells is accumulative. Different cell lines differ in their responses when exposed to glutamate. In this review article the responses of PC12, SH-SY5Y, HT-22, NT-2, OLCs, C6, primary rat cortical neurons, RGC-5, and SCN2.2 cell systems are systematically collected and analyzed.