Immunojanus Particles for low-volume and isolation-free unlabeled characterization of small Extracellular Vesicle in biofluids: Characterization of disease type by surface marker profiling.

Small extracellular vesicles (sEVs) are vital for cellular communication and serve as critical biomarker carriers for diseases such as cancer. However, quantifying and profiling sEV surface markers presents significant challenges due to the low concentration of specific sEV-bound proteins and interference by more abundant dispersed proteins. This paper presents Immunojanus Particles (IJPs), a new method that enables the direct detection of sEVs in less than an hour without isolation. The design of IJPs incorporates fluorescent and non-fluorescent halves, utilizing rotational Brownian motion to detect captured sEVs through the change in the blinking rate, without interference from the smaller dispersed proteins. We demonstrate a detection limit of 2E5 sEVs/mL with low sample volumes and the capability to characterize sEVs directly from plasma, serum, cell culture media, and urine. In a small pilot study involving 87 subjects, including individuals with colorectal cancer, pancreatic ductal adenocarcinoma, glioblastoma, Alzheimer's disease, and healthy controls, our method accurately identified the type of disease with high 0.90-0.99 AUC in a blind setting. Compared with an orthogonal ultracentrifugation plus surface plasmon resonance (UC+SPR) method that requires about 24 hours, the sensitivity and dynamic range of IJP are better by 2 logs.

Extracellular vesicle and lipoprotein diagnostics (ExoLP-Dx) with membrane sensor: A robust microfluidic platform to overcome heterogeneity.

The physiological origins and functions of extracellular vesicles (EVs) and lipoproteins (LPs) propel advancements in precision medicine by offering non-invasive diagnostic and therapeutic prospects for cancers, cardiovascular, and neurodegenerative diseases. However, EV/LP diagnostics (ExoLP-Dx) face considerable challenges. Their intrinsic heterogeneity, spanning biogenesis pathways, surface protein composition, and concentration metrics complicate traditional diagnostic approaches. Commonly used methods such as nanoparticle tracking analysis, enzyme-linked immunosorbent assay, and nuclear magnetic resonance do not provide any information about their proteomic subfractions, including active proteins/enzymes involved in essential pathways/functions. Size constraints limit the efficacy of flow cytometry for small EVs and LPs, while ultracentrifugation isolation is hampered by co-elution with non-target entities. In this perspective, we propose a charge-based electrokinetic membrane sensor, with silica nanoparticle reporters providing salient features, that can overcome the interference, long incubation time, sensitivity, and normalization issues of ExoLP-Dx from raw plasma without needing sample pretreatment/isolation. A universal EV/LP standard curve is obtained despite their heterogeneities.

An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma.

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.

Theoretical Design and Synthesis of Caged Compounds Using X-Ray-Triggered Azo Bond Cleavage.

Caged compounds are frequently used in life science research. However, the light used to activate them is commonly absorbed and scattered by biological materials, limiting their use to basic research in cells or small animals. In contrast, hard X-rays exhibit high bio-permeability due to the difficulty of interacting with biological molecules. With the main goal of developing X-ray activatable caged compounds, azo compounds are designed and synthesized with a positive charge and long π-conjugated system to increase the reaction efficiency with hydrated electrons. The azo bonds in the designed compounds are selectively cleaved by X-ray, and the fluorescent substance Diethyl Rhodamine is released. Based on the results of experiments and quantum chemical calculations, azo bond cleavage is assumed to occur via a two-step process: a two-electron reduction of the azo bond followed by N─N bond cleavage. Cellular experiments also demonstrate that the azo bonds can be cleaved intracellularly. Thus, caged compounds that can be activated by an azo bond cleavage reaction promoted by X-ray are successfully generated.

Whole exome sequencing identifies novel variants of PIK3CA and validation of hotspot mutation by droplet digital PCR in breast cancer among Indian population.

BACKGROUND: Breast cancer (BC) is the most common malignancy with very high incidence and relatively high mortality in women. The PIK3CA gene plays a pivotal role in the pathogenicity of breast cancer. Despite this, the mutational status of all exons except exons 9 and 20 still remains unknown. METHODS: This study uses the whole exome sequencing (WES) based approach to identify somatic PIK3CA mutations in Indian BC cohorts. The resultant hotspot mutations were validated by droplet digital PCR (ddPCR). Further, molecular dynamics (MD) simulation was applied to elucidate the conformational and functional effects of hotspot position on PIK3CA protein. RESULTS: In our cohort, PIK3CA showed a 44.4% somatic mutation rate and was among the top mutated genes. The mutations of PIK3CA were confined in Exons 5, 9, 11, 18, and 20, whereas the maximum number of mutations lies within exons 9 and 20. A total of 9 variants were found in our study, of which 2 were novel mutations observed on exons 9 (p.H554L) and 11 (p.S629P). However, H1047R was the hotspot mutation at exon 20 (20%). In tumor tissues, there was a considerable difference between copy number of wild-type and H1047R mutant was detected by ddPCR. Significant structural and conformational changes were observed during MD simulation, induced due to point mutation at H1047R/L position. CONCLUSIONS: The current study provides a comprehensive view of novel as well as reported single nucleotide variants (SNVs) in PIK3CA gene associated with Indian breast cancer cases. The mutation status of H1047R/L could serve as a prognostic value in terms of selecting targeted therapy in BC.

Outcomes of three-piece rigid scleral fixated intraocular lens implantation in subjects with deficient posterior capsule following complications in manual small incision cataract surgery.

Objective: To evaluate the surgical visual outcomes of three-piece rigid scleral fixated intraocular lens (SFIOL) implantation in subjects with deficient posterior capsule following complications of cataract extraction. Design: Retrospective 4-year cohort study. Participants: Data from 174 eyes that underwent SFIOL combined with pars plana vitrectomy (PPV) between January 2018 and March 2022 and follow-up exams were included. Methods: Demographic characteristics including primary indications for surgery, history of trauma, laterality, baseline and best-corrected visual acuity (BCVA), refraction as spherical equivalent (SE), intraocular pressure (IOP), duration of follow-up, and complications were analyzed. Results: The mean preoperative BCVA was 1.38 ± 0.46 logarithm of the minimum angle of resolution (logMAR), which improved significantly to 0.37 ± 0.22 logMAR. The baseline refractive status measured in spherical equivalent (SE) was 4.1 ± 6.2 Diopters (D), and the postoperative status was -0.4 ± 0.97 D. Early postoperative complications included hypotony (n = 1; 0.57%, vitreous hemorrhage (n = 3; 1.72%), elevated IOP (n = 8; 4.59%), mild dilated pupil (n = 1; 0.57%) and corneal edema (n = 16; 9.19%). Late complications included in this study were retinal detachment (n = 1; 0.57%), cystoid macular edema (CME) (n = 1; 0.57%), primary glaucoma (n = 1; 0.57%), secondary glaucoma (n = 13; 7.47%), zonular dehiscence (n = 3; 1.72%), retinal pigment epithelium (RPE) changes (n = 3; 1.72%), choroidal coloboma (n = 2; 1.14%), posterior dislocation of posterior chamber IOL (PCIOL) (n = 1; 0.57%), corneal decompensation (n = 1; 0.57%), retinal hemorrhage (n = 1; 0.57%), macular hole (n = 1; 0.57%), chronic uveitis (n = 1; 0.57%), mild non-proliferative diabetic retinopathy (NPDR) (n = 3; 1.72%), and mild NPDR with diabetic macular edema (DME) (n = 1; 0.57%). Conclusion: Integrating IOL implantation with vitrectomy various posterior segment complications were resolved in the same setting without attempting a second surgery.

Detection of EGFR and its Activity State in Plasma CD63-EVs from Glioblastoma Patients: Rapid Profiling using an Anion Exchange Membrane Sensor.

We present a novel quantitative immunoassay for CD63 EVs (extracellular vesicles) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane and a charged silica nanoparticle reporter. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins and fluorophore degradation, thus enabling direct plasma analysis. With a limit of detection of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. Glioblastoma necessitates improved non-invasive diagnostic approaches for early detection and monitoring. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. This approach offers direct glioblastoma detection from untreated human patient samples. Analysis of glioblastoma clinical samples yielded an area-under-the-curve (AUC) value of 0.99 and low p-value of 0.000033, significantly surpassing the performance of existing assays and markers.

Assessments of TP53 and CTNNB1 gene hotspot mutations in circulating tumour DNA of hepatitis B virus-induced hepatocellular carcinoma.

Background: Hepatitis B virus (HBV) infection is one of the major causes of chronic liver disease, which progresses from chronic hepatitis B (CHB) to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Early detection and laboratory-based screening of hepatocellular carcinoma are still major challenges. This study was undertaken to determine whether the cancer hallmark gene signatures that are released into circulation as circulating tumour DNA (ctDNA) can be used as a liquid biopsy marker for screening, early detection, and prognosis of HCC. Methods: A total of 130 subjects, including HBV-HCC (n = 80), HBV-cirrhotic and non-cirrhotic (n = 35), and healthy (n = 15) controls, were evaluated for TP53 and beta-catenin (CTNNB1) gene hotspot mutations in ctDNA by Sanger-based cycle sequencing and droplet digital PCR (ddPCR) assays. Mutation detection frequency, percentage mutant fractions, and their association with tumour stage, mortality, and smoking habits were determined. Results: Sanger-based cycle sequencing was carried out for 32 HCC patients. Predict SNP Tools analysis indicated several pathogenic driver mutations in the ctDNA sequence, which include p.D228N, p.C229R, p.H233R, p.Y234D, p.S240T, p.G245S, and p.R249M for TP53 gene exon 7 and p.S33T for CTNNB1 gene exon 3. The TP53 c.746G>T (p.R249M) mutation was detected predominately (25% cases) by sequencing, but there was no dominant mutation at position c.747G>T (p.R249S) that was reported for HBV-HCC patients. A dual-probe ddPCR assay was developed to determine mutant and wild-type copy numbers of TP53 (p.R249M and p.R249S) and CTNNB1 (p.S45P) and their percentage mutant fraction in all 130 subjects. The TP53 R249M and CTNNB1 S45P mutations were detected in 31.25% and 26.25% of HCC patients, respectively, with a high mutant-to-wild-type fraction percentage (1.81% and 1.73%), which is significant as compared to cirrhotic and non-cirrhotic patients. Poor survival was observed in HCC patients with combined TP53 and CTNNB1 gene driver mutations. The TP53 R249M mutation was also significantly (p < 0.0001) associated with smoking habits (OR, 11.77; 95% CI, 3.219-36.20), but not the same for the TP53 R249S mutation. Conclusion: Screening of ctDNA TP53 and CTNNB1 gene mutations by ddPCR may be helpful for early detection and identifying the risk of HCC progression.

Disentangling the enigmatic role of ephrin signaling in chronic pain: Moving towards future anti-pain therapeutics.

Chronic pain is a common and debilitating condition with a huge social and economic burden worldwide. Currently, available drugs in clinics are not adequately effective and possess a variety of severe side effects leading to treatment withdrawal and poor quality of life. The ongoing search for new therapeutics with minimal side effects for chronic pain management remains a high research priority. Erythropoietin-producing human hepatocellular carcinoma cell receptor (Eph) is a tyrosine kinase receptor that is involved in neurodegenerative disorders, including pain. The Eph receptor interacts with several molecular switches, such as N methyl d-aspartate receptor (NMDAR), mitogen-activated protein kinase (MAPK), calpain 1, caspase 3, protein kinase a (PKA), and protein kinase Cy (PKCy), which in turn regulates pathophysiology of chronic pain. Here we highlight the emerging evidence of the Ephs/ephrin system as a possible near-future therapeutic target for the treatment of chronic pain and discuss the various mechanism of its involvement. We critically analyse the present status of Eph receptor system and conclude that extrapolating the pharmacological and genetic approaches using a strong therapeutic development framework could serve as next-generation analgesics for the management of chronic pain.

Below-threshold harmonic generation in gas-jets for Th-229 nuclear spectroscopy.

The generation of below-threshold harmonics in gas-jets constitutes a promising path towards optical frequency combs in the vacuum ultra-violet (VUV) spectral range. Of particular interest is the 150 nm range, which can be exploited to probe the nuclear isomeric transition of the Thorium-229 isotope. Using widely available high-power, high-repetition-rate Ytterbium-based laser sources, VUV frequency combs can be generated through the process of below-threshold harmonic generation, in particular 7th harmonic generation of 1030 nm. Knowledge about the achievable efficiencies of the harmonic generation process is crucial for the development of suitable VUV sources. In this work, we measure the total output pulse energies and conversion efficiencies of below-threshold harmonics in gas-jets in a phase-mismatched generation scheme using Argon and Krypton as nonlinear media. Using a 220 fs, 1030 nm source, we reach a maximum conversion efficiency of 1.1 × 10-5 for the 7th harmonic (147 nm) and 0.78 × 10-4 for the 5th harmonic (206 nm). In addition, we characterize the 3rd harmonic of a 178 fs, 515 nm source with a maximum efficiency of 0.3%.

High glucose-induced increasing reactive nitrogen species accumulation triggered mitochondrial dysfunction, inflammation, and apoptosis in keratinocytes.

Growing evidence indicates that skin injuries are a common complication of diabetes. However, the cellular and molecular mechanisms of high glucose (HG) environment trigger nitrosative stress-mediated inflammation and apoptosis in keratinocytes remains unknown. Here we investigated whether reactive nitrogen species (RNS) induced by HG environment restrain antioxidant activity, and mitochondrial dysfunction leading to inflammation, and apoptosis via stress signaling pathways in keratinocytes. Our results established that the HG environment enhanced the production of nitric oxide (NO) and peroxynitrite anion (ONOO-) by inducible NO synthase (iNOS) in keratinocytes. Overproduction of RNS in HG environment suppress the antioxidants activity leading to mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, decrease in mitochondrial transcription factor A(TFAM), increase in mitochondrial DNA (mtDNA) displacement loop (D-loop) and decrease in glycolytic flux concentration, which was attenuated by pharmacological inhibitors of NO/ONOO-, Nω-Nitro-l-argininemethyl ester hydrochloride (NAME)/hydralazine hydrochloride (Hyd.HCl). Excess production of RNS in HG environment restrained 8-oxoguanine DNA glycosylase-1 (OGG1) expression and increased 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA were regulated by NO or ONOO-. Further, HG-induced RNA production caused an increase in the production of inflammatory mediators accompanied by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascade, lipid peroxidation (LPO), and protein carbonylation (PC) reactions followed by breakdown the cell-cell communication and apoptosis. Pre-treatment of cell with NAME/Hyd.HCl, diminished the expression of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3, inflammatory mediators, and attenuated apoptosis in keratinocytes. Together, our results indicated that excess production of RNS in HG environment triggered inflammation and apoptosis, mediated by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascades in keratinocytes.

Evaluation of the cell-free DNA integrity index as a liquid biopsy marker to differentiate hepatocellular carcinoma from chronic liver disease.

Background: Hepatocellular carcinoma (HCC) occurs in the majority of patients with underlying chronic liver disease (CLD) of viral and non-viral etiologies, which requires screening for early HCC diagnosis. Liquid biopsy holds great promise now for early detection, prognosis, and assessment of response to cancer therapy. Cell-free DNA (cfDNA) as a liquid biopsy marker can be easily detected by a real-time quantitative PCR (RT-qPCR) assay for a change in its concentration, integrity, and fragmentation in cancer. Methods: Patients with HCC (n = 100), CLD (n = 100), and healthy (n = 30) controls were included in the study. The cfDNA was isolated from serum and real-time quantitative PCR (RT-qPCR) was carried out using primer pairs for large (>205 bp) and small (110 bp) fragments of repetitive elements (ALU and LINE1) and housekeeping genes (ß-Actin and GAPDH). Total cfDNA concentrations and integrity index were determined by the absolute quantitation method (L/S ratio or cfDII-integrity). The cfDII as a measure of fragmentation was determined by comparative Ct (2-ΔΔCt) method of relative quantification (cfDII-fragmentation). Using a receiver operating characteristic (ROC) curve, cfDII-integrity and cfDII-fragmentation were used to differentiate HCC from CLD patients or healthy controls. Results: The total cfDNA concentrations in the sera of HCC (244 ng/ml) patients were significantly higher than those of CLD (33 ng/ml) patients and healthy (16.88 ng/ml) controls. HCC patients have shown poor DNA integrity or excess cfDNA fragmentation than CLD patients and healthy controls. The cfDII-integrity of GAPDH and ALU fragment significantly differentiate HCC from CLD at AUROC 0.72 and 0.67, respectively. The cfDII-fragmentation following normalization with cfDNA of healthy control has shown significant differential capabilities of HCC from CLD at AUROC 0.67 using GAPDH and 0.68 using the ALU element. The ROC curve of LINE1 and ß-actin cfDII was not found significant for any of the above methods. The cfDII-fragmentation trend in HCC patients of different etiologies was similar indicating increased cfDNA fragmentation irrespective of its etiology. Conclusion: The cfDII measuring both DNA integrity (L/S ratio) and fragmentation of the Alu and GAPDH genes can differentiate HCC from CLD patients and healthy individuals.

In Vitro Anticancer Activity of Methanolic Extract of Justicia adhatoda Leaves with Special Emphasis on Human Breast Cancer Cell Line.

Natural products are being targeted as alternative anticancer agents due to their non-toxic and safe nature. The present study was conducted to explore the in vitro anticancer potential of Justicia adhatoda (J. adhatoda) leaf extract. The methanolic leaf extract was prepared, and the phytochemicals and antioxidant potential were determined by LCMS analysis and DPPH radical scavenging assay, respectively. A docking study performed with five major alkaloidal phytoconstituents showed that they had a good binding affinity towards the active site of NF-κB. Cell viability assay was carried out in five different cell lines, and the extract exhibited the highest cytotoxicity in MCF-7, a breast cancer cell line. Extract-treated cells showed a significant increase in nitric oxide and reactive oxygen species production. Cell cycle analysis showed an arrest in cell growth at the Sub-G0 phase. The extract successfully inhibited cell migration and colony formation and altered mitochondrial membrane potential. The activities of superoxide dismutase and glutathione were also found to decrease in a dose-dependent manner. The percentage of apoptotic cells was found to increase in a dose-dependent manner in MCF-7 cells. The expressions of caspase-3, Bax, and cleaved-PARP were increased in extract-treated cells. An increase in the expression of NF-κB was found in the cytoplasm in extract-treated cells. J. adhatoda leaf extract showed a potential anticancer effect in MCF-7 cells.

Structural Characterization of Nickel-Doped Aluminum Oxide Cations by Cryogenic Ion Trap Vibrational Spectroscopy.

Isolated nickel-doped aluminum oxide cations (NiOm)(Al2O3)n(AlO)+ with m = 1-2 and n = 1-3 are investigated by infrared photodissociation (IRPD) spectroscopy in combination with density functional theory and the single-component artificial force-induced reaction method. IRPD spectra of the corresponding He-tagged cations are reported in the 400-1200 cm-1 spectral range and assigned based on a comparison to calculated harmonic IR spectra of low-energy isomers. Simulated spectra of the lowest energy structures generally match the experimental spectra, but multiple isomers may contribute to the spectra of the m = 2 series. The identified structures of the oxides (m = 1) correspond to inserting a Ni-O moiety into an Al-O bond of the corresponding (Al2O3)1-3(AlO)+ cluster, yielding either a doubly or triply coordinated Ni2+ center. The m = 2 clusters prefer similar structures in which the additional O atom either is incorporated into a peroxide unit, leaving the oxidation state of the Ni2+ atom unchanged, or forms a biradical comprising a terminal oxygen radical anion Al-O•- and a Ni3+ species. These clusters represent model systems for under-coordinated Ni sites in alumina-supported Ni catalysts and should prove helpful in disentangling the mechanism of selective oxidative dehydrogenation of alkanes by Ni-doped catalysts.

Optically superior fluorescent probes for selective imaging of cells, tumors, and reactive chemical species.

Fluorescent chemical probes have become powerful tools to study biological events in living cells. They provide a great opportunity to quantitatively and qualitatively analyze the physiological and biochemical properties of living cells in real time. The ability of researchers to manipulate these probes for a desired specific purpose has turned many heads in the scientific community. Despite a slow start, fluorescent probe research has seen exponential growth over the last decade in the world. This change required some adventurous and creative scientists from different fields-like biology, medicine, and chemistry-to come together to facilitate the constant expansion of this field. This review article introduces some fundamental concepts related to fluorescent probe designing and development. It also summarizes various fluorescent probes with superior optical properties used in fields like cell biology, cellular imaging, medical research, and cancer diagnosis. It is hoped that this article will encourage more young and creative scientists to contribute their talents to this field.

Real-world re-treatment outcomes of direct-acting antiviral therapy failure in patients with chronic hepatitis C.

Direct-acting antiviral (DAA) drugs are associated with high (>95%) sustained virological response at 12 weeks (SVR12) in chronic hepatitis C (CHC) patients. There is a paucity of data regarding the characteristics and re-treatment outcomes of DAA treatment failure patients. In a retrospective analysis of the prospectively collected database, we assessed the outcomes of re-treatment among patients with previous DAA failure. Patients' characteristics, viral characteristics, including resistance-associated substitutions (RAS) in a subgroup of patients, SVR12, and clinical outcomes were studied. Of 40 patients with DAA failure, among whom 36 were retreated, mean age was 45.7 years, 63.9% (n = 23) were male, 63.9% (n = 23) had a genotype-3 infection and 63.9% (n = 23) were cirrhotic. The re-treatment regimens included a combination of pan-genotypic DAA, mainly sofosbuvir and velpatasvir with or without ribavirin. Three patients who declined retreatment and one who was still on treatment was excluded. For patients who completed re-treatment, SVR12 was 100% irrespective of genotypes. SVR12 among genotype 3 was 75% (15 of 20) when lost to follow-up was considered a treatment failure. Six patients died due to liver-related causes, including five (83.3%) with hepatocellular carcinoma. RAS analysis in 17 randomly selected patients did not reveal any dominant substitutions in NS5A or NS5B region affecting SVR12, though several novel mutations were observed. In conclusion, re-treatment of CHC patients with prior DAA failure using pan-genotypic DAA is associated with high SVR12 rates irrespective of genotype or the presence of RAS.

Prognostic and Therapeutic Potentials of OncomiRs Modulating mTOR Pathways in Virus-Associated Hepatocellular Carcinoma.

BACKGROUND: Dysregulated oncomiRs are attributed to hepatocellular carcinoma (HCC) through targeting mTOR signaling pathway responsible for cell growth and proliferation. The potential of these oncomiRs as biomarker for tumor response or as target for therapy needs to be evaluated. AIM: Tumor response assessment by OncomiR changes following locoregional therapy (LRT) and targeting of these oncomiRs modulating pathway. METHODS: All consecutive viral-HCC patients of BCLC stage-A/B undergoing LRT were included. OncomiRs (miR-21, -221, and -16) change in circulation and AFP-ratio at 1-month post-LRT to baseline was estimated to differentiate various categories of response as per mRECIST criteria. OncomiR modulating mTOR pathway was studied by generating miR-21 and miR-221 overexpressing Huh7 stable cell lines. RESULTS: Post-LRT tumor response was assessed in 90 viral-HCC patients (CR, 40%; PR, 31%, and PD, 29%). Significant increase of miRNA-21 and -221 expression was observed in PD (p = 0.040, 0.047) and PR patients (miR-21, p = 0.045). Fold changes of miR-21 can differentiate response in group (CR from PR+PD) at AUROC 0.718 (95% CI, 0.572-0.799) and CR from PD at AUROC 0.734 (95% CI, 0.595-0.873). Overexpression of miR-21 in hepatoma cell line had shown increased phosphorylation p70S6K, the downstream regulator of cell proliferation in mTOR pathway. Upregulation of AKT, mTOR, and RPS6KB1 genes were found significant (P < 0.005) and anti-miR-21 specifically reduced mTOR gene (P = 0.02) expression. CONCLUSIONS: The miR-21 fold change correlates well with imaging in predicting tumor response. Overexpression of miR-21 has a role in HCC through mTOR pathway activation and can be targeted.

Regioselective 6-endo-dig iodocyclization: an accessible approach for iodo-benzo[a]phenazines.

A facile approach for the synthesis of substituted iodo-benzo[a]phenazines from 2-aryl-3-(aryl/alkylethynyl)quinoxalines via 6-endo-dig ring closure has been described under mild reaction conditions. Iodocyclization proceeds through the iodonium ion intermediate followed by nucleophilic cyclization with the C-H bond of the arene. Furthermore, the resulting 6-iodo-5-aryl/alkyl benzo[a]phenazine derivatives allowed for structural diversification by employing various coupling reactions. The structure of iodo-benzo[a]phenazine was confirmed by X-ray crystallographic studies of the compound.

Regio- and Stereoselective Domino Synthesis of Oxazolo Fused Pyridoindoles and Benzofurooxazolo Pyridines from ortho-Alkynylarylaldehydes.

An environmentally benign Au(III)-catalyzed regio- and stereoselective domino synthesis of oxazolo fused pyridoindoles 7a-v and benzofurooxazolo pyridines 8a-n by the reaction of o-alkynylaldehydes 4a-t and 5a-k with (S)-phenylglycinol 6a and (R)-phenylglycinol 6b under mild reaction conditions using water as reaction medium is reported. The reaction proceeded via selective C-N bond formation on the more electrophilic alkynyl carbon through 6-endo-dig cyclization. The reaction tolerates a wide variety of functional groups. The developed chemistry has been successfully extended for the synthesis of a diverse class of γ-carbolines and benzofuro[3,2-c]pyridines using corresponding ester hydrochlorides of serine, threonine, and cystine as a nitrogen source.

High-Throughput Multiplexed Peptide-Centric Profiling Illustrates Both Substrate Cleavage Redundancy and Specificity in the MMP Family.

Matrix metalloproteinases (MMPs) play incompletely understood roles in health and disease. Knowing the MMP cleavage preferences is essential for a better understanding of the MMP functions and design of selective inhibitors. To elucidate the cleavage preferences of MMPs, we employed a high-throughput multiplexed peptide-centric profiling technology involving the cleavage of 18,583 peptides by 18 proteinases from the main sub-groups of the MMP family. Our results enabled comparison of the MMP substrates on a global scale, leading to the most efficient and selective substrates. The data validated the accuracy of our cleavage prediction software. This software allows us and others to locate, with nearly 100% accuracy, the MMP cleavage sites in the peptide sequences. In addition to increasing our understanding of both the selectivity and the redundancy of the MMP family, our study generated a roadmap for the subsequent MMP structural-functional studies and efficient substrate and inhibitor design.