Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Marker-assisted introgression to improve the oleic acid content in the TMV 7 groundnut (Arachis hypogaea L.) variety suitable for the oil industry.

BACKGROUND: Improving the quality and shelf life of groundnut oil is one of the foremost objectives of groundnut breeding programmes. This can be achieved by marker-assisted introgression, a technique that efficiently and precisely enables breeders to develop plants with enhanced qualities. This study focused on improving the oleic acid content of an elite groundnut variety, TMV 7, by introgressing a recessive mutation responsible for the increase in oleic acid from ICG 15419. Hybridization was performed between the donor and recurrent parents to develop the F1, BC1F1, BC2F1 and BC2F2 populations. Introgressed lines with increased oleic acid in the genetic background of TMV 7 were identified using allele-specific marker, F435-F, F435SUB-R and a set of SSR markers were employed to recover the genome of the recurrent parent. RESULTS: With two backcrosses, a total of ten homozygous plants in the BC2F2 population were identified with oleic acid content ranging from 54.23 to 57.72% causing an increase of 36% over the recurrent parent. Among the ten lines, the line IL-23 exhibited the highest level of recurrent parent genome recovery of 91.12%. CONCLUSIONS: The phenotypic evaluation of 10 homozygous introgressed lines indicated fewer differences for all other traits under study compared to the recurrent parent, except for oleic acid and linoleic acid content confirming the genetic background of the recurrent parent. The identified lines will be subjected to multilocation trials before their commercial release.

High mortality in free-ranging wild boars associated with African swine fever virus in India.

The present study focuses on the pathological and molecular characterization of African swine fever virus (ASFV) associated with an outbreak in wild boars in two national parks in southern India in 2022-2023. Significant mortality was observed among free-ranging wild boars at Bandipur National Park, Karnataka, and Mudumalai National Park, Tamil Nadu. Extensive combing operations were undertaken in both national parks, spanning an area of around 100 km2, originating from the reported epicenter, to estimate the mortality rate. Recovered carcasses were pathologically examined, and ASFV isolates was genetically characterized. Our findings suggested spillover infection of ASFV from nearby domestic pigs, and the virus was equally pathogenic in wild boars and domestic pigs. ASFV intrusion was reported in the Northeastern region of the country, which borders China and Myanmar, whereas the current outbreak is very distantly located, in southern India. Molecular data will help in tracing the spread of the virus in the country.

Retrospective database analysis for clinical diagnoses commonly associated with pneumococcal diseases in the Malaysian healthcare system over a 3-year period (2013-2015).

BACKGROUND: Pneumococcal disease caused by Streptococcus pneumoniae is an important cause of morbidity and mortality across all ages, particularly in younger children and older adults. Here, we describe pneumococcal disease hospitalizations at Ministry of Health (MoH) facilities in Malaysia between 2013 and 2015. METHODS: This was a retrospective databases analysis. Tabular data from the Malaysian Health Data Warehouse (MyHDW) were used to identify microbiologically confirmed, pneumococcal disease hospitalizations and deaths during hospitalization, using hospital-assigned ICD-10 codes (i.e., classified as meningitis, pneumonia, or non-meningitis non-pneumonia). Case counts, mortality counts, and case fatality rates were reported by patient age group and by Malaysian geographic region. RESULTS: A total of 683 pneumococcal disease hospitalizations were identified from the analysis: 53 pneumococcal meningitis hospitalizations (5 deaths and 48 discharges), 413 pneumococcal pneumonia hospitalizations (24 deaths and 389 discharges), and 205 non-meningitis non-pneumonia pneumococcal disease hospitalizations (58 deaths and 147 discharges). Most hospitalizations occurred in children aged < 2 years. Crude mortality was highest among children aged < 2 years (for all three disease categories), among adults aged ≥ 65 years (for pneumococcal pneumonia), or among adults aged 65-85 years (for non-meningitis non-pneumonia pneumococcal disease). The case fatality rate, all ages included, was 5.8% for pneumococcal pneumonia, 9.1% for pneumococcal meningitis, and 28.3% for non-meningitis non-pneumonia pneumococcal disease. CONCLUSIONS: Our study is the first to document pneumococcal disease hospitalizations and deaths during hospitalization in Malaysia. Although this database analysis likely underestimated case counts, and the true disease burden could be even greater, the study demonstrates a substantial burden of pneumococcal disease. Public health measures, including vaccination, would significantly contribute to the prevention of hospitalizations and deaths associated with pneumococcal disease in Malaysia.

Clinico-immunological evaluation of use of omega-3 fatty acids as nutraceutical approach in management of patients with chronic periodontitis: A randomized clinical trial.

Background: Subgingival bacterial colonization and biofilm formation are known to be the main etiology of periodontal disease progression. This biofilm elicits host response and the interaction between host defence mechanisms with plaque microorganisms and their products results in periodontal disease. Host modulatory therapy (HMT) is a form of treatment of periodontitis that focuses on treatment of the host in the host-bacteria interaction. Omega-3 fatty acids have emerged as a potential HMT agent to treat inflammation associated with periodontal disease. Methods: A total of 60 cases of chronic periodontitis were allocated into two groups; the test group (n = 30) were treated with scaling and root planing (SRP) and given a dietary supplementation of omega-3 fatty acid while the control group were treated with SRP alone. Clinical parameters carried out were plaque index (PI), gingival bleeding index (GBI), pocket probing depth (PPD) and clinical attachment level (CAL) and immunological parameter included interleukin-1ß level in saliva at baseline, 3 months and 6 months after therapy. Results: At 6 months, both the groups showed significant improvements with regards to all clinical and immunological parameters compared to baseline (all p < 0.05). However, test group presented with more favourable statistically significant results. Conclusion: The use of omega-3 fatty acid as nutraceutical agent to conventional method acted as beneficial therapeutic measures and effective in patients with chronic periodontitis when compared with SRP alone.

Hemin-Induced Death Models Hemorrhagic Stroke and Is a Variant of Classical Neuronal Ferroptosis.

Ferroptosis is a caspase-independent, iron-dependent form of regulated necrosis extant in traumatic brain injury, Huntington disease, and hemorrhagic stroke. It can be activated by cystine deprivation leading to glutathione depletion, the insufficiency of the antioxidant glutathione peroxidase-4, and the hemolysis products hemoglobin and hemin. A cardinal feature of ferroptosis is extracellular signal-regulated kinase (ERK)1/2 activation culminating in its translocation to the nucleus. We have previously confirmed that the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor U0126 inhibits persistent ERK1/2 phosphorylation and ferroptosis. Here, we show that hemin exposure, a model of secondary injury in brain hemorrhage and ferroptosis, activated ERK1/2 in mouse neurons. Accordingly, MEK inhibitor U0126 protected against hemin-induced ferroptosis. Unexpectedly, U0126 prevented hemin-induced ferroptosis independent of its ability to inhibit ERK1/2 signaling. In contrast to classical ferroptosis in neurons or cancer cells, chemically diverse inhibitors of MEK did not block hemin-induced ferroptosis, nor did the forced expression of the ERK-selective MAP kinase phosphatase (MKP)3. We conclude that hemin or hemoglobin-induced ferroptosis, unlike glutathione depletion, is ERK1/2-independent. Together with recent studies, our findings suggest the existence of a novel subtype of neuronal ferroptosis relevant to bleeding in the brain that is 5-lipoxygenase-dependent, ERK-independent, and transcription-independent. Remarkably, our unbiased phosphoproteome analysis revealed dramatic differences in phosphorylation induced by two ferroptosis subtypes. As U0126 also reduced cell death and improved functional recovery after hemorrhagic stroke in male mice, our analysis also provides a template on which to build a search for U0126's effects in a variant of neuronal ferroptosis.SIGNIFICANCE STATEMENT Ferroptosis is an iron-dependent mechanism of regulated necrosis that has been linked to hemorrhagic stroke. Common features of ferroptotic death induced by diverse stimuli are the depletion of the antioxidant glutathione, production of lipoxygenase-dependent reactive lipids, sensitivity to iron chelation, and persistent activation of extracellular signal-regulated kinase (ERK) signaling. Unlike classical ferroptosis induced in neurons or cancer cells, here we show that ferroptosis induced by hemin is ERK-independent. Paradoxically, the canonical MAP kinase kinase (MEK) inhibitor U0126 blocks brain hemorrhage-induced death. Altogether, these data suggest that a variant of ferroptosis is unleashed in hemorrhagic stroke. We present the first, unbiased phosphoproteomic analysis of ferroptosis as a template on which to understand distinct paths to cell death that meet the definition of ferroptosis.

Temporary induction of hypoxic adaptations by preconditioning fails to enhance Schwann cell transplant survival after spinal cord injury.

Hypoxic preconditioning is protective in multiple models of injury and disease, but whether it is beneficial for cells transplanted into sites of spinal cord injury (SCI) is largely unexplored. In this study, we analyzed whether hypoxia-related preconditioning protected Schwann cells (SCs) transplanted into the contused thoracic rat spinal cord. Hypoxic preconditioning was induced in SCs prior to transplantation by exposure to either low oxygen (1% O2 ) or pharmacological agents (deferoxamine or adaptaquin). All preconditioning approaches induced hypoxic adaptations, including increased expression of HIF-1α and its target genes. These adaptations, however, were transient and resolved within 24 h of transplantation. Pharmacological preconditioning attenuated spinal cord oxidative stress and enhanced transplant vascularization, but it did not improve either transplanted cell survival or recovery of sensory or motor function. Together, these experiments show that hypoxia-related preconditioning is ineffective at augmenting either cell survival or the functional outcomes of SC-SCI transplants. They also reveal that the benefits of hypoxia-related adaptations induced by preconditioning for cell transplant therapies are not universal.

Characterization of infectious laryngotracheitis virus isolates from laying hens during 2019-2020 outbreaks in Tamil Nadu, India.

Infectious laryngotracheitis (ILT) is an acute respiratory disease in chickens that is a serious threat to poultry-producing countries worldwide. In the present study, we isolated and characterized infectious laryngotracheitis (ILTV) virus isolates by sequencing and restriction fragment length polymorphism analysis of PCR-amplified products (PCR-RFLP). A total of 26 ILTV outbreaks were investigated that occurred between 2019 and 2020 in flocks that had not been vaccinated against ILTV. ILTV was isolated by cultivating tracheal samples in embryonated chicken eggs, which showed multiple opaque pock lesions and thickening of the chorioallantoic membrane after 120 hours of infection. The ILTV isolates were identified and characterized by PCR and sequencing a portion of the ICP4 and TK genes. Phylogenetic analysis based on the ICP4 region showed that the sequences clustered with chicken-embryo-origin vaccine-like strains. Sequence analysis of the ICP4 region differentiated chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and field ILTV strains, with significant differences in nucleotide and amino acid sequences. Furthermore, PCR-RFLP analysis of the TK gene showed that the patterns were identical to those obtained with low-virulence and vaccine strains. In conclusion, sequencing of a portion of the ICP4 region of ILTV allowed differentiation of ILTV field, CEO, and TCO vaccine strains. In this study, CEO-vaccine-like strains were found to be the cause of ILTV outbreaks between 2019 and 2020 in Tamil Nadu in southern India.

Induction of antiviral and cell mediated immune responses significantly reduce viral load in an acute foot-and-mouth disease virus infection in cattle.

Foot-and-mouth disease virus (FMDV) causes a severe infection in ruminant animals. Here we present an in-depth transcriptional analysis of soft-palate tissue from cattle experimentally infected with FMDV. The differentially expressed genes from two Indian cattle (Bos indicus) breeds (Malnad Gidda and Hallikar) and Holstein Friesian (HF) crossbred calves, highlighted the activation of metabolic processes, mitochondrial functions and significant enrichment of innate antiviral immune response pathways in the indigenous calves. The results of RT-qPCR based validation of 12 genes was in alignment with the transcriptome data. The indigenous calves showing lesser virus load, elicited early neutralizing antibodies and IFN-γ immune responses. This study revealed that induction of potent innate antiviral response and cell mediated immunity in indigenous cattle, especially Malnad Gidda, significantly restricted FMDV replication during acute infection. These data highlighting the molecular processes associated with host-pathogen interactions, could aid in the conception of novel strategies to prevent and control FMDV infection in cattle.

Seroepidemiology revealed neosporosis as an under-realised entity in dairy cattle reared in South India.

From the dairy herds (n = 16) reared in few localities of South India with the history of reproductive inefficiency and incidental abortion, 176 sera samples from Jersey (n = 108) and Holstein Friesian (n = 68) crossbred cows were collected to detect prevalence of bovine neosporosis antibodies induced by Neospora caninum (N. caninum) through competitive enzyme linked immuno-sorbent assay (cELISA). The overall true prevalence was found as 23.5% whereas 7.7, 19.1, 25.7 and 40.5% was observed in cows of less than 1 year, 1 to 3 years, 3 to 6 years and above 6 years of age, respectively, denoting that increase in prevalence of N. caninum antibody correlated directly with the age. Among the cattle with and without abortion, 41.1 and 20.6% of true prevalence was found, respectively. The breed-wise true prevalence was 24.3 and 22.2% in Jersey and Holstein Friesians crossbred cows, respectively, indicating that crossbred cows of both breeds are equally susceptible to neosporosis. The prevalence of N. caninum antibody might be attributed to coexistence of dogs resulting in contamination of feed with dog faeces. The presence of dogs with the cattle herd predisposed the herd 3.59 times more to acquire neosporosis than the herd without dogs. The annual estimated economic loss in an aborted herd of having 11 animals was 0.23 million INR due to loss of both calf and milk yield.

Epigenetic regulators of neuronal ferroptosis identify novel therapeutics for neurological diseases: HDACs, transglutaminases, and HIF prolyl hydroxylases.

A major thrust of our laboratory has been to identify how physiological stress is transduced into transcriptional responses that feed back to overcome the inciting stress or its consequences, thereby fostering survival and repair. To this end, we have adopted the use of an in vitro model of ferroptosis, a caspase-independent, but iron-dependent form of cell death (Dixon et al., 2012; Ratan, 2020). In this review, we highlight three distinct epigenetic targets that have evolved from our studies and which have been validated in vivo studies. In the first section, we discuss our studies of broad, pan-selective histone deacetylase (HDAC) inhibitors in ferroptosis and how these studies led to the validation of HDAC inhibitors as candidate therapeutics in a host of disease models. In the second section, we discuss our studies that revealed a role for transglutaminase as an epigenetic modulator of proferroptotic pathways and how these studies set the stage for recent elucidation of monoamines as post-translation modifiers of histone function. In the final section, we discuss our studies of iron-, 2-oxoglutarate-, and oxygen-dependent dioxygenases and the role of one family of these enzymes, the HIF prolyl hydroxylases, in mediating transcriptional events necessary for ferroptosis in vitro and for dysfunction in a host of neurological conditions. Overall, our studies highlight the importance of epigenetic proteins in mediating prodeath and prosurvival responses to ferroptosis. Pharmacological agents that target these epigenetic proteins are showing robust beneficial effects in diverse rodent models of stroke, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

The drug adaptaquin blocks ATF4/CHOP-dependent pro-death Trib3 induction and protects in cellular and mouse models of Parkinson's disease.

Identifying disease-causing pathways and drugs that target them in Parkinson's disease (PD) has remained challenging. We uncovered a PD-relevant pathway in which the stress-regulated heterodimeric transcription complex CHOP/ATF4 induces the neuron prodeath protein Trib3 that in turn depletes the neuronal survival protein Parkin. Here we sought to determine whether the drug adaptaquin, which inhibits ATF4-dependent transcription, could suppress Trib3 induction and neuronal death in cellular and animal models of PD. Neuronal PC12 cells and ventral midbrain dopaminergic neurons were assessed in vitro for survival, transcription factor levels and Trib3 or Parkin expression after exposure to 6-hydroxydopamine or 1-methyl-4-phenylpyridinium with or without adaptaquin co-treatment. 6-hydroxydopamine injection into the medial forebrain bundle was used to examine the effects of systemic adaptaquin on signaling, substantia nigra dopaminergic neuron survival and striatal projections as well as motor behavior. In both culture and animal models, adaptaquin suppressed elevation of ATF4 and/or CHOP and induction of Trib3 in response to 1-methyl-4-phenylpyridinium and/or 6-hydroxydopamine. In culture, adaptaquin preserved Parkin levels, provided neuroprotection and preserved morphology. In the mouse model, adaptaquin treatment enhanced survival of dopaminergic neurons and substantially protected their striatal projections. It also significantly enhanced retention of nigrostriatal function. These findings define a novel pharmacological approach involving the drug adaptaquin, a selective modulator of hypoxic adaptation, for suppressing Parkin loss and neurodegeneration in toxin models of PD. As adaptaquin possesses an oxyquinoline backbone with known safety in humans, these findings provide a firm rationale for advancing it towards clinical evaluation in PD.

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

OBJECTIVES: N-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke. METHODS: Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC. RESULTS: NAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ). INTERPRETATION: NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.

An Enhancement of Deep Learning Algorithm for Brain Tumor Segmentation Using Kernel Based CNN with M-SVM.

The brain tumor can be created by uncontrollable increase of abnormal cells in tissue of brain and it has two kinds of tumors: one is benign and another one is malignant tumor. The benign brain tumor does not affect the adjacent normal and healthy tissue but the malignant tumor can affect the neighboring tissues of brain that can lead to the death of person. An early detection of brain tumor can be required to protect the survival of patients. Usually, the brain tumor is detected using MRI scanning method. However, the radiologists are not providing the effective tumor segmentation in MRI image due to the irregular shape of tumors and position of tumor in the brain. Accurate brain tumor segmentation is needed to locate the tumor and it is used to give the correct treatment for a patient and it provides the key to the doctor who must execute the surgery for patient. In this paper, a novel deep learning algorithm (kernel based CNN) with M-SVM is presented to segment the tumor automatically and efficiently. This presented work contains several steps that are preprocessing, feature extraction, image classification and tumor segmentation of brain. The MRI image is smoothed and enhanced by Laplacian of Gaussian filtering method (LoG) with Contrast Limited Adaptive Histrogram Equalization (CLAHE) and feature can be extracted from it based on tumor shape position, shape and surface features in brain. Consequently, the image classification is done using M-SVM depending on the selected features. From MRI image, the tumor is segmented with help of kernel based CNN method.. Experimental results of proposed method can show that this presented technique can executes brain tumor segmentation accurately reaching almost 84% in evaluation with existing algorithms.

Occult HBV infection in HIV-infected adults and evaluation of pooled NAT for HBV.

The study aimed to determine the prevalence of occult hepatitis B virus infection among HIV-infected persons and to evaluate the use of a pooling strategy to detect occult HBV infection in the setting of HIV infection. Five hundred and two HIV-positive individuals were tested for HBV, occult HBV and hepatitis C and D with serologic and nucleic acid testing (NAT). We also evaluated a pooled NAT strategy for screening occult HBV infection among the HIV-positive individuals. The prevalence of HBV infection among HIV-positive individuals was 32 (6.4%), and occult HBV prevalence was 10%. The pooling HBV NAT had a sensitivity of 66.7% and specificity of 100%, compared to HBV DNA NAT of individual samples. In conclusion, this study found a high prevalence of occult HBV infection among our HIV-infected population. We also demonstrated that pooled HBV NAT is highly specific, moderately sensitive and cost-effective. As conventional HBV viral load assays are expensive in resource-limited settings such as India, pooled HBV DNA NAT might be a good way for detecting occult HBV infection and will reduce HBV-associated complications.

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage leads to disability or death with few established treatments. Adverse outcomes after intracerebral hemorrhage result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. METHODS: We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were used. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. RESULTS: Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy, or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased after intracerebral hemorrhage in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by >80% and had similar therapeutic windows in vitro. CONCLUSIONS: Experimental intracerebral hemorrhage shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either of these pathways can bring cells below that threshold to survival.

Hydroxamic acid-based histone deacetylase (HDAC) inhibitors can mediate neuroprotection independent of HDAC inhibition.

Histone deacetylase (HDAC) inhibition improves function and extends survival in rodent models of a host of neurological conditions, including stroke, and neurodegenerative diseases. Our understanding, however, of the contribution of individual HDAC isoforms to neuronal death is limited. In this study, we used selective chemical probes to assess the individual roles of the Class I HDAC isoforms in protecting Mus musculus primary cortical neurons from oxidative death. We demonstrated that the selective HDAC8 inhibitor PCI-34051 is a potent neuroprotective agent; and by taking advantage of both pharmacological and genetic tools, we established that HDAC8 is not critically involved in PCI-34051's mechanism of action. We used BRD3811, an inactive ortholog of PCI-34051, and showed that, despite its inability to inhibit HDAC8, it exhibits robust neuroprotective properties. Furthermore, molecular deletion of HDAC8 proved insufficient to protect neurons from oxidative death, whereas both PCI-34051 and BRD3811 were able to protect neurons derived from HDAC8 knock-out mice. Finally, we designed and synthesized two new, orthogonal negative control compounds, BRD9715 and BRD8461, which lack the hydroxamic acid motif and showed that they stably penetrate cell membranes but are not neuroprotective. These results indicate that the protective effects of these hydroxamic acid-containing small molecules are likely unrelated to direct epigenetic regulation via HDAC inhibition, but rather due to their ability to bind metals. Our results suggest that hydroxamic acid-based HDAC inhibitors may mediate neuroprotection via HDAC-independent mechanisms and affirm the need for careful structure-activity relationship studies when using pharmacological approaches.

Nanohydroxyapatite-reinforced chitosan composite hydrogel for bone tissue repair in vitro and in vivo.

BACKGROUND: Bone loss during trauma, surgeries, and tumor resection often results in critical-sized bone defects that need to be filled with substitutionary materials. Complications associated with conventional grafting techniques have led to the development of bioactive tissue-engineered bone scaffolds. The potential application of hydrogels as three-dimensional (3D) matrices in tissue engineering has gained attention in recent years because of the superior sensitivity, injectability, and minimal invasive properties of hydrogels. Improvements in the bioactivity and mechanical strength of hydrogels can be achieved with the addition of ceramics. Based on the features required for bone regeneration, an injectable thermosensitive hydrogel containing zinc-doped chitosan/nanohydroxyapatite/beta-glycerophosphate (Zn-CS/nHAp/ß-GP) was prepared and characterized, and the effect of nHAp on the hydrogel was examined. METHODS: Hydrogels (Zn-CS/ß-GP, Zn-CS/nHAp/ß-GP) were prepared using the sol-gel method. Characterization was carried out by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) as well as swelling, protein adsorption, and exogenous biomineralization studies. Expression of osteoblast marker genes was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analyses. In vivo bone formation was studied using a rat bone defect model system. RESULTS: The hydrogels exhibited sol-gel transition at 37°C. The presence of nHAp in the Zn-CS/nHAp/ß-GP hydrogel enhanced swelling, protein adsorption, and exogenous biomineralization. The hydrogel was found to be non-toxic to mesenchymal stem cells. The addition of nHAp to the hydrogel also enhanced osteoblast differentiation under osteogenic conditions in vitro and accelerated bone formation in vivo as seen from the depositions of apatite and collagen. CONCLUSIONS: The synthesized injectable hydrogel (Zn-CS/nHAp/ß-GP) showed its potential toward bone formation at molecular and cellular levels in vitro and in vivo. The current findings demonstrate the importance of adding nHAp to the hydrogel, thereby accelerating potential clinical application toward bone regeneration.

Bioassay guided fractionation and identification of active anti-inflammatory constituent from Delonix elata flowers using RAW 264.7 cells.

CONTEXT: Delonix elata (L.) Gamble (Fabaceae) has been used in the Indian traditional medicine system to treat rheumatism and inflammation. AIM: To assess the anti-inflammatory effect of Delonix elata flowers and to isolate the active principle. MATERIALS AND METHODS: The prompt anti-inflammatory constituent was isolated from Delonix elata flower extracts using bioassay guided fractionation in liposaccharide (LPS) stimulated RAW 264.7 macrophage cell line. The anti-inflammatory activity of extracts/fractions/sub-fractions/compounds (10, 25, and 50 µg/ml) was evaluated by estimating the levels of nitric oxide (NO), TNF-α, and IL-1ß after 24 h of LPS induction (1 µg/ml). The isolated active compound was subjected to NMR, IR, and UV analyses for structure determination. RESULTS: In an attempt to search for anti-inflammatory constituents, the active pure principle was isolated and crystallized as a white compound from Delonix elata flowers methanol extract. This active compound (50 µg/ml) decreased the release of inflammatory mediators levels such as NO (0.263 ± 0.03 µM), TNFα (160.20 ± 17.57 pg/ml), and IL-1ß (285.79 ± 15.16 pg/ml) significantly (p < 0.05); when compared to the levels of NO (0.774 ± 0.08 µM), TNFα (501.71 ± 25.14 pg/ml), and IL-1ß (712.68 ± 52.25 pg/ml) from LPS-stimulated macrophage cells. The active compound was confirmed as hesperidin with NMR, IR, and UV spectroscopy data. This is the first report of this compound from Delonix elata flowers. CONCLUSION: The findings of the study support the traditional use of Delonix elata flowers to treat inflammation.

Swertiamarin ameliorates inflammation and osteoclastogenesis intermediates in IL-1ß induced rat fibroblast-like synoviocytes.

OBJECTIVE AND DESIGN: Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1ß induced fibroblast-like synoviocytes (FLS). METHODS: The FLS were isolated from Freund's Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1ß. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10-50 µg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h). RESULTS: IL-1ß induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03%) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22%) and NO production (82.06 ± 3.91% at 50 µg/ml) in a dose-dependent manner. It also significantly (P < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly (P < 0.05) attenuated the release of the same in time dependent mode. CONCLUSION: These findings suggest that treatment with swertiamarin attenuated IL-1ß induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.