Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia.

Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL). Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells. Transplantation of polyclonal Ikaros-mutant pre-B cells resulted in long-latency oligoclonal pre-B-ALL, which demonstrates that loss of Ikaros contributes to multistep B cell leukemogenesis. Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL.

CircAMOTL1 RNA and AMOTL1 Protein: Complex Functions of AMOTL1 Gene Products.

The complexity of the cellular proteome facilitates the control of a wide range of cellular processes. Non-coding RNAs, including microRNAs and long non-coding RNAs, greatly contribute to the repertoire of tools used by cells to orchestrate various functions. Circular RNAs (circRNAs) constitute a specific class of non-coding RNAs that have recently emerged as a widely generated class of molecules produced from many eukaryotic genes that play essential roles in regulating cellular processes in health and disease. This review summarizes current knowledge about circRNAs and focuses on the functions of AMOTL1 circRNAs and AMOTL1 protein. Both products from the AMOTL1 gene have well-known functions in physiology, cancer, and other disorders. Using AMOTL1 as an example, we illustrate how focusing on both circRNAs and proteins produced from the same gene contributes to a better understanding of gene functions.

Nematode Genome Announcement: A Draft Genome of Seed Gall Nematode, Anguina tritici.

Anguina tritici is the first plant-parasitic nematode described in literature, dating back to the year 1743. It is responsible for causing earcockle (seed gall) and tundu diseases in wheat and rye. Notably, this nematode has been observed to survive in an anhydrobiotic state for up to 32 years within wheat seed galls. These exceptional characteristics have inspired the sequencing of the A. tritici genome. In this study, we present the initial draft genome of A. tritici, obtained using the Illumina MiSeq platform with coverage of 60-fold. The genome is estimated to have a size of 164 Mb and comprises 39,965 protein-coding genes, exhibiting a GC content of 39.1%. The availability of this genome data will serve as a foundation for future functional biological investigations, particularly for genes whose functions remain unknown to this day.

Immune Immunomodulation in Coronavirus Disease 2019 (COVID-19): Strategic Considerations for Personalized Therapeutic Intervention.

We are learning that the host response to severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2) infection is complex and highly dynamic. Effective initial host defense in the lung is associated with mild symptoms and disease resolution. Viral evasion of the immune response can lead to refractory alveolar damage, ineffective lung repair mechanisms, and systemic inflammation with associated organ dysfunction. The immune response in these patients is highly variable and can include moderate to severe systemic inflammation and/or marked systemic immune suppression. There is unlikely to be a "one size fits all" approach to immunomodulation in patients with coronavirus disease 2019 (COVID-19). We believe that a personalized, immunophenotype-driven approach to immunomodulation that may include anticytokine therapy in carefully selected patients and immunostimulatory therapies in others is the shortest path to success in the study and treatment of patients with critical illness due to COVID-19.

Host-delivered RNAi-mediated silencing of the root-knot nematode (Meloidogyne incognita) effector genes, Mi-msp10 and Mi-msp23, confers resistance in Arabidopsis and impairs reproductive ability of the root-knot nematode.

MAIN CONCLUSION: Mi-msp10 and Mi-msp23 effector genes play a significant role during Meloidogyne incognita parasitism on Arabidopsis roots. The role of these genes was confirmed by demonstrating the decrease of the level of susceptibility of Arabidopsis by the  silencing of Mi-msp10 and Mi-msp23 genes using HD-RNAi technology. Root-knot nematodes (RKNs) are the most damaging pathogens severely affecting global food production. The sustainable options to minimize menace of nematode populations through economically feasible measures are limited. Thus, the development of innovative and target-specific strategies that aid in their management is imperative. RNAi technology has emerged as a sustainable and target-specific alternative to control phytonematodes. Here, we characterized two novel subventral gland and dorsal gland-specific effectors, Mi-msp10 and Mi-msp23, to determine their potential effectiveness in controlling M. incognita. Comparative developmental profiling using qRT-PCR revealed higher expression of both effectors in the adult nematode female. Furthermore, functional evaluation of Mi-msp10 and Mi-msp23 dsRNA cassettes was performed using host-delivered RNAi (HD-RNAi) in Arabidopsis. The transgenic lines were examined against M. incognita, and the phenotypic effect of HD-RNAi was evident with a 61% and 51% reduction in gall formation in the Mi-msp10 and Mi-msp23 RNAi lines, respectively. A significant drop in the nematode adult females by 59% for Mi-msp10 and 49% for Mi-msp23-RNAi lines was observed. Similarly, production in egg masses decreased significantly by 76% (Mi-msp10) and 60% (Mi-msp23) for the RNAi lines, which eventually decreased the reproductive factor by 92% and 75%, respectively. The gene expression analysis showed a significant decrease in the transcript level by up to 72% (Mi-msp10) and 66% (Mi-msp23) in M. incognita females feeding on RNAi lines, providing further evidence of effective gene silencing. Overall, our findings provide useful information and support further development of RNAi-based strategies to control M. incognita.

Anti-inflammatory potential of novel hexapeptide derived from Meretrix meretrix foot and its functional properties.

The study aimed to identify bioactive peptide from Meretrix meretrix Linnaeus foot (MMF) and examine its potential of suppressing inflammation. In brief, the anti-inflammatory activity was identified by erythrocyte membrane protection and protein denaturation assay from MMF peptic 9th-h hydrolysate and was separated with three molecular weight cut-off units. The obtained four fractions were testified for activity and the fraction (10-3 kDa) with maximum activity was purified using gel permeation chromatography. Finally, the peptide sequence was identified as Asn-Pro-Ala-Gln-Asp-Cys (647.559 Da) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hexapeptide was characterised for functional properties at different pH range. The non-toxic hexapeptide was able to reduce the cyclooxygenase (COX)-2 activation, pro-inflammatory cytokines and nitric oxide (NO) production significantly in RAW264.7 macrophage cells. The current results propose that the hexapeptide derived from MMF protein can act as an effective anti-inflammatory against pro-inflammatory cytokines, COX-2 and NO. Moreover, it could be used as an effective alternative source for drugs in pharma and also as an ingredient in food industries.

Effect of NaCl-Induced Salinity Stress on Growth, Osmolytes and Enzyme Activities in Wheat Genotypes.

Effect of NaCl-induced salinity stress was studied on three wheat (Triticum aestivum L.) genotypes (Platinum Lok 1, Eagle 135 and Kisan farmer). Seedling growth characteristics, chlorophyll contents, lipid peroxidation (MDA contents), proline, glycine betaine (GB) and the activities of catalase and peroxidase enzymes were evaluated at the seedling stage. The magnitude of reduction in growth parameters and chlorophyll contents was less in Platinum Lok 1 as compared to Kisan Farmer and Eagle 135. Platinum Lok 1 exhibited the highest increase in free proline and GB contents under NaCl stress as compared to the other two genotypes. Compared to Platinum Lok 1 and Eagle 135, Kisan Farmer accumulated more MDA contents under salinity stress. The enzyme activities were significantly higher in Platinum Lok 1 when compared to the other two genotypes. Therefore, these parameters could provide useful markers for the identification of salinity stress tolerant wheat genotypes at the seedling stage.

Neuroprotective effects of urate are mediated by augmenting astrocytic glutathione synthesis and release.

Urate has emerged as a promising target for neuroprotection based on epidemiological observations, preclinical models, and early clinical trial results in multiple neurologic diseases, including Parkinson's disease (PD). This study investigates the astrocytic mechanism of urate's neuroprotective effect. Targeted biochemical screens of conditioned medium from urate- versus vehicle-treated astrocytes identified markedly elevated glutathione (GSH) concentrations as a candidate mediator of urate's astrocyte-dependent neuroprotective effects. Urate treatment also induced the nuclear translocation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and transcriptional activation of its key target genes in primary astrocytic cultures. Urate's neuroprotective effect was attenuated when GSH was depleted in the conditioned media either by targeting its synthesis or release by astrocytes. Overall, these results implicate GSH as the extracellular astrocytic factor mediating the protective effect of urate in a cellular model of PD. These results also show that urate can employ a novel indirect neuroprotective mechanism via induction of the Nrf2 signaling pathway, a master regulator of the response to oxidative stress, in astrocytes.

Oral Administration of a Specific p300/CBP Lysine Acetyltransferase Activator Induces Synaptic Plasticity and Repairs Spinal Cord Injury.

TTK21 is a small-molecule activator of p300/creb binding protein (CBP) acetyltransferase activity, which, upon conjugation with a glucose-derived carbon nanosphere (CSP), can efficiently cross the blood-brain barrier and activate histone acetylation in the brain. Its role in adult neurogenesis and retention of long-term spatial memory following intraperitoneal (IP) administration is well established. In this study, we successfully demonstrate that CSP-TTK21 can be effectively administered via oral gavage. Using a combination of molecular biology, microscopy, and electrophysiological techniques, we systematically investigate the comparative efficacy of oral administration of CSP and CSP-TTK21 in wild-type mice and evaluate their functional effects in comparison to intraperitoneal (IP) administration. Our findings indicate that CSP-TTK21, when administered orally, induces long-term potentiation in the hippocampus without significantly altering basal synaptic transmission, a response comparable to that achieved through IP injection. Remarkably, in a spinal cord injury model, oral administration of CSP-TTK21 exhibits efficacy equivalent to that of IP administration. Furthermore, our research demonstrates that oral delivery of CSP-TTK21 leads to improvements in motor function, histone acetylation dynamics, and increased expression of regeneration-associated genes (RAGs) in a spinal injury rat model, mirroring the effectiveness of IP administration. Importantly, no toxic and mutagenic effects of CSP-TTK21 are observed at a maximum tolerated dose of 1 g/kg in Sprague-Dawley (SD) rats via the oral route. Collectively, these results underscore the potential utility of CSP as an oral drug delivery system, particularly for targeting the neural system.

Utility of monocyte HLA-DR and rationale for therapeutic GM-CSF in sepsis immunoparalysis.

Sepsis, a heterogeneous clinical syndrome, features a systemic inflammatory response to tissue injury or infection, followed by a state of reduced immune responsiveness. Measurable alterations occur in both the innate and adaptive immune systems. Immunoparalysis, an immunosuppressed state, associates with worsened outcomes, including multiple organ dysfunction syndrome, secondary infections, and increased mortality. Multiple immune markers to identify sepsis immunoparalysis have been proposed, and some might offer clinical utility. Sepsis immunoparalysis is characterized by reduced lymphocyte numbers and downregulation of class II human leukocyte antigens (HLA) on innate immune monocytes. Class II HLA proteins present peptide antigens for recognition by and activation of antigen-specific T lymphocytes. One monocyte class II protein, mHLA-DR, can be measured by flow cytometry. Downregulated mHLA-DR indicates reduced monocyte responsiveness, as measured by ex-vivo cytokine production in response to endotoxin stimulation. Our literature survey reveals low mHLA-DR expression on peripheral blood monocytes correlates with increased risks for infection and death. For mHLA-DR, 15,000 antibodies/cell appears clinically acceptable as the lower limit of immunocompetence. Values less than 15,000 antibodies/cell are correlated with sepsis severity; and values at or less than 8000 antibodies/cell are identified as severe immunoparalysis. Several experimental immunotherapies have been evaluated for reversal of sepsis immunoparalysis. In particular, sargramostim, a recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF), has demonstrated clinical benefit by reducing hospitalization duration and lowering secondary infection risk. Lowered infection risk correlates with increased mHLA-DR expression on peripheral blood monocytes in these patients. Although mHLA-DR has shown promising utility for identifying sepsis immunoparalysis, absence of a standardized, analytically validated method has thus far prevented widespread adoption. A clinically useful approach for patient inclusion and identification of clinically correlated output parameters could address the persistent high unmet medical need for effective targeted therapies in sepsis.

Evaluation of Peri-Implant Parameters and C-Reactive Protein Levels among Patients with Different Obesity Levels.

Background: Assessment of correlation between peri-implant parameters and C-reactive protein levels among patients with different obesity levels. Materials and Methods: Evaluation of 60 subjects was performed who were scheduled to undergo dental implant therapy for missing mandibular first molars. Three study cohorts were formed, namely, Group A: obese group (BMI between 30 Kg/m2 and 34.9 Kg/m2), Group B: high obese group (BMI over 34.9 Kg/m2), and Group C: non-obese group (BMI under 25 Kg/m2). Each cohort comprised 20 subjects. Dental implant therapy was carried out in all the patients. Peri-implant variables were evaluated in all the patients. Blood samples were obtained, and C-reactive protein levels in subjects having different obesity levels. Statistical analysis was performed using SPSS software. Results: Mean serum C-reactive protein levels among patients of groups A, B, and C occurred to be 3.28 mg/L, 3.65 g/L, and 3.61 g/L, respectively. On comparing numerically, noticeable outcomes were achieved. Mean probing depth among subjects of groups A, B, and C occurred to be 2.9 mm, 3.2 mm, and 1.3 mm, respectively. Mean marginal bone loss among subjects of groups A, B, and C occurred to be 2.1 mm, 2.7 mm, and 0.8 mm, respectively. On comparing numerically, noteworthy outcomes were gathered. Conclusion: There were significantly higher deranged peri-implant inflammatory variables among patients with higher levels of obesity.

Gall-specific promoter, an alternative to the constitutive CaMV35S promoter, drives host-derived RNA interference targeting Mi-msp2 gene to confer effective nematode resistance.

One of the major obligate plant parasites causing massive economic crop losses belongs to the class of root-knot nematodes (RKNs). Targeting of major nematode parasitism genes via Host Delivered-RNAi (HD-RNAi) to confer silencing is established as one of the most effective approaches to curb nematode infection. Utilizing nematode-responsive root-specific (NRRS) promoters to design a dsRNA molecule targeting approach to hamper nematode parasitism. Here, a previously validated peroxidase gall specific promoter, pAt2g18140, from Arabidopsis was employed to express the dsRNA construct of the nematode effector gene Mi-msp2 from Meloidogyne incognita. Arabidopsis RNAi lines of CaMV35S::Mi-msp2-RNAi and pAt2g18140::Mi-msp2-RNAi were compared with control plants to assess the decrease in plant nematode infection. When subjected to infection, the maximum reductions in the numbers of galls, females and egg masses in the CaMV35S::Mi-msp2-RNAi lines were 61%, 66% and 95%, respectively, whereas for the pAt2g18140::Mi-msp2-RNAi lines, they were 63%, 68% and 100%, respectively. The reduction in transcript level ranged from 79%-82% for CaMV35S::Mi-msp2-RNAi and 72%-79% for the pAt2g18140::Mi-msp2-RNAi lines. Additionally, a reduction in female size and a subsequent reduction in next-generation fecundity demonstrate the efficacy and potential of the gall specific promoter pAt2g18140 for utilization in the development of HD-RNAi constructs against RKN, as an excellent alternative to the CaMV35S promoter.

Glucose derived carbon nanosphere (CSP) conjugated TTK21, an activator of the histone acetyltransferases CBP/p300, ameliorates amyloid-beta 1-42 induced deficits in plasticity and associativity in hippocampal CA1 pyramidal neurons.

The master epigenetic regulator lysine acetyltransferase (KAT) p300/CBP plays a pivotal role in neuroplasticity and cognitive functions. Recent evidence has shown that in several neurodegenerative diseases, including Alzheimer's disease (AD), the expression level and function of p300/CBP are severely compromised, leading to altered gene expression causing pathological conditions. Here, we show that p300/CBP activation by a small-molecule TTK21, conjugated to carbon nanosphere (CSP) ameliorates Aß-impaired long-term potentiation (LTP) induced by high-frequency stimulation, theta burst stimulation, and synaptic tagging/capture (STC). This functional rescue was correlated with CSP-TTK21-induced changes in transcription and translation. Mechanistically, we observed that the expression of a large number of synaptic plasticity- and memory-related genes was rescued, presumably by the restoration of p300/CBP mediated acetylation. Collectively, these results suggest that small-molecule activators of p300/CBP could be a potential therapeutic molecule for neurodegenerative diseases like AD.

Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases.

Notch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G(1)-S progression of cell cycle in T cells. Here we show that expression of the G(1) proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from gamma-secretase inhibitor (GSI)-induced G(1) arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.

Angiotensin I-Converting Enzyme (ACE-I) Inhibition and Antioxidant Peptide from a Squilla Species.

BACKGROUND: Oratosquilla woodmasoni is one of the marine squilla species, which is found in the entire Asia-Pacific region. This current study assesses the species as the main basis of both ACEi and antioxidant peptide. OBJECTIVE: To isolate the ACEi peptide derived from O. woodmasoni and examine its ACE inhibition along with antioxidant potential. MATERIALS AND METHODS: The squilla muscle protein was hydrolysed using alcalase and trypsin enzymes for 12 hours and tested for DH. The hydrolysates were examined for their ACEi activity and then the best hydrolysate was sequentially purified in various chromatographical methods. The purified peptide was studied for anti-oxidant and functional properties, followed by amino acid sequencing. The purified peptide was also evaluated for its toxicity by in vitro cell viability assay. RESULTS: The DH% was found to be 47.13 ± 0.72% and 89.43 ± 2.06% for alcalase and trypsin, respectively. The alcalase 5th-hour hydrolysate was detected with potent activity (65.97 ± 0.56%) using ACEi assay and was primarily fractionated using ultrafiltration; the maximum inhibitory activity was found with 77.04 ± 0.52% in 3-10 kDa fraction. Subsequently, the fraction was purified using IEC and GFC, in which the AC1-A2 fraction had higher antihypertensive activity (70.85 ± 0.78%). The non-toxic fraction showed hexapeptide HVGGCG with molecular weight 529 Da with great potential of antioxidant activity along with functional property. CONCLUSION: This peptide could be developed as a potential ACE-inhibitory and antioxidant agent.

A Meretrix meretrix visceral mass derived peptide inhibits lipopolysaccharide-stimulated responses in RAW264.7 cells and adult zebrafish model.

The Meretrix meretrix is abundantly present in the Indian coastal areas which can be used as an important useful bioactive source for industrial applications. The M. meretrix visceral mass (MMV) was hydrolysed with four different enzymes and verified for anti-inflammatory activity with the help of HRBC membrane stabilization (HMS) and albumin denaturation (AD) assay. Among the hydrolysates, the tryptic 6th hour hydrolysate was selected for purification using ultrafiltration and size-exclusion chromatography (SEC). Further, the purified peptide was identified to have six amino acid sequence (HKGQCC, 675.582 Da). However, to confirm the anti-inflammatory effects of the purified peptide, it was investigated for nitric oxide synthase (iNOS), pro-inflammatory cytokines production as well as cyclooxygenase-2 (COX-2) activation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and also evaluated for its functional properties. The in-vitro gastrointestinal digestion was performed on the peptide which cleaved the peptide into two i.e. MMV1 (HK, 284.1 Da) and MMV2 (GQCC, 410.1 Da). The data suggested that the MMV2 peptide have maximum activity and was found to be stable at high temperatures. The MMV2 peptide demonstrated abrupt localization throughout the adult zebrafish body and successfully downregulated the mRNA levels of inflammation-related genes in LPS-induced adult zebrafish. This study indicates that the peptide MMV2 possesses anti-inflammatory activity by suppressing the induced inflammation and can be a strong competitor against non-steroidal anti-inflammatory drugs (NSAIDs).

Sargramostim and immune checkpoint inhibitors: combinatorial therapeutic studies in metastatic melanoma.

The use of immune checkpoint inhibitors in patients with metastatic melanoma generates clinical benefit, including improved survival. Yet disease resistance and immune-related adverse events persist as unmet needs. Sargramostim, a yeast-derived recombinant human GM-CSF, has shown clinical activity against diverse solid tumors, including metastatic melanoma. Here we review the use of sargramostim for treatment of advanced melanoma. Potential sargramostim applications in melanoma draw on the unique ability of GM-CSF to link innate and adaptive immune responses. We review preclinical and translational data describing the mechanism of action of sargramostim and synergy with immune checkpoint inhibitors to enhance efficacy and reduce treatment-related toxicity.

Lay abstract Immune checkpoint inhibitors are medications that help the immune system to fight cancer. Side effects with these medicines may occur because the immune system may attack healthy cells. Sargramostim is a medication that is similar to a protein in the body (GM-CSF). Studies have shown that sargramostim can fight cancer, including melanoma. When sargramostim is used with immune checkpoint inhibitors, the body's natural defense to fight cancer (the immune system) is boosted and some side effects are reduced. This article reviews how GM-CSF is thought to boost the immune system's response against cancer in the laboratory and in animal models. We also review the use of sargramostim alone and combined with ipilimumab in patients with advanced melanoma.

EGLLGDVF: A Novel Peptide from Green Mussel Perna viridis Foot Exerts Stability and Anti-inflammatory Effects on LPS-Stimulated RAW264.7 Cells.

BACKGROUND: Green mussel Perna viridis is a bivalve mollusc which is native to the Indian coast and can be found in the Indo-Pacific as well as Asia-Pacific regions. This study evaluates the P. viridis foot (PVF) as a source of an anti-inflammatory peptide. OBJECTIVE: To characterize and evaluate the possibility of pro-inflammatory cytokines, nitric oxide (NO) as well as cyclooxygenase (COX)-2 reduction in RAW264.7 cells and to analyze functional aspects of the derived peptide from PVF. MATERIALS AND METHODS: The PVF was hydrolysed with different enzymes and the antiinflammatory activity of hour hydrolysates were evaluated using HRBC Membrane Stabilization (HMS) against hypotonicity induced haemolysis and Albumin Denaturation (AD) inhibition from induced heat assays. Later, the active hour hydrolysate was separated by ultrafiltration and purified using Size-Exclusion Chromatography (SEC). Further, the purified peptide's sequence was identified using LC-MS/MS and functional properties were determined. Also, the peptide was observed for its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 cells for pro-inflammatory cytokines, NO production and COX-2 activation. RESULTS: Among the four enzymes 6th hour alcalase hydrolysate exhibited potent anti-inflammatory activity and was sequentially fractioned with molecular weight cut-offs; further active fraction (30- 10 kDa) was purified. The active peak-II was identified as EGLLGDVF (849.435 Da) and exhibited decent functional aspects. The peptide successfully reduced the production of pro-inflammatory cytokines, NO and COX-2 activation; and down-regulated the iNOS and COX-2 protein expression in LPS-stimulated RAW264.7 cells. CONCLUSION: Our study indicates that EGLLGDVF derived from PVF has potential antiinflammatory applications applicable in food and pharmaceutical industries.

Aberrant pain modulation in trigeminal neuralgia patients.

Objectives The present study attempts to understand the role of supraspinal nociceptive pain modulation in typical trigeminal neuralgia (TN) patients by using a conditioned pain modulation paradigm and estimation of plasma levels of two important neuromodulators; Calcitonin Gene-Related Peptide and ß-endorphin. Methods Twenty TN patients and 20 healthy, age and gender-matched subjects participated in the study. The participants' hot pain thresholds (HPT) were measured over their affected side on the face. Testing sites were matched for healthy controls. For the conditioned pain modulation their contralateral foot was immersed in noxious cold (5 °C) water bath (conditioning stimuli) for 30 s and HPT (testing stimuli) was determined before, during and till 5 min after the immersion. Plasma Calcitonin Gene-Related Peptide and ß-endorphin levels were estimated to understand their role in disease pathogenesis and pain modulation. Results Change in HPT during foot immersion was significantly higher in healthy controls compared to TN patients (p<0.0001). The changes recorded in HPT in patients, were significant only in 2nd and 3rd minute post immersion. While in healthy controls, the effect lasted till the 4th minute. The concentration of beta-endorphin was significantly lower in TN patients (p=0.003) when compared to healthy controls. Conclusions The results suggest that there is an impairment in supraspinal pain modulation also known as Diffuse Noxious Inhibitory Controls in typical TN and that the reduced levels of ß-endorphin may contribute to the chronic pain state experienced by patients.