Detection of feline immunodeficiency virus by neutral red-based loop-mediated isothermal amplification assay.

Background and Aim: Feline immunodeficiency virus (FIV) is a retroviral pathogen globally responsible for immunodeficiency disease in cats. However, the current diagnosis based on antibody detection has limitations and can also produce false-positive results. This study aimed to develop a one-pot loop-mediated isothermal amplification (LAMP) process integrated with neutral red (NR-LAMP) assay for detection of FIV proviral DNA. Materials and Methods: We developed a one-pot, gag gene-based NR-LAMP for convenient, rapid, specific, and sensitive colorimetric inspection of FIV proviral DNA. Results: The developed NR-LAMP was capable of amplifying at an optimum temperature of 65°C for 40 min. No cross-amplification was detected between FIV and other feline viruses tested, indicating the high specificity (98.44%) of the novel FIV-LAMP primer. Our NR-LAMP assay has a detection limit of 4.2 × 101 copies/µL. A total of 80 clinical samples with a background of FIV infection were collected and tested using the proposed method. The NR-LAMP assay showed a high sensitivity of 100% compared to conventional polymerase chain reaction assay. Conclusion: These results support the suitability of NR-LAMP as a potential future alternative clinical molecular approach for further use in the diagnosis of FIV-infected cats.

Exploring the apoptotic effects of sericin on HCT116 cells through comprehensive nanostring transcriptomics and proteomics analysis.

Sericin, a silk protein from Bombyx mori (silkworms), has many applications, including cosmetics, anti-inflammation, and anti-cancer. Sericin complexes with nanoparticles have shown promise for breast cancer cell lines. Apoptosis, a programmed cell death mechanism, stops cancer cell growth. This study found that Sericin urea extract significantly affected HCT116 cell viability (IC50 = 42.00 ± 0.002 µg/mL) and caused apoptosis in over 80% of treated cells. S-FTIR analysis showed significant changes in Sericin-treated cells' macromolecule composition, particularly in the lipid and nucleic acid areas, indicating major cellular modifications. A transcriptomics study found upregulation of the apoptotic signaling genes FASLG, TNFSF10, CASP3, CASP7, CASP8, and CASP10. Early apoptotic proteins also showed that BAD, AKT, CASP9, p53, and CASP8 were significantly upregulated. A proteomics study illuminated Sericin-treated cells' altered protein patterns. Our results show that Sericin activated the extrinsic apoptosis pathway via the caspase cascade (CASP8/10 and CASP3/7) and the death receptor pathway, involving TNFSF10 or FASLG, in HCT116 cells. Upregulation of p53 increases CASP8, which activates CASP3 and causes HCT116 cell death. This multi-omics study illuminates the molecular mechanisms of Sericin-induced apoptosis, sheds light on its potential cancer treatment applications, and helps us understand the complex relationship between silk-derived proteins and cellular processes.

Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase.

The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06-2 µM and 0.15-2 µM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.

In Vitro Evaluation of Antidiabetic Potential of Cleistocalyx nervosum var. paniala Fruit Extract.

Diabetes mellitus is a complex global public health condition. Medicinal plants are significant resources in the research of alternative new drug active compounds. Cleistocalyx nervosum var. paniala (C. nervosum) is an indigenous berry fruit widely grown in Southeast Asia. The fruit of C. nervosum exhibit various medicinal properties and health benefits. This study aimed to investigate antidiabetic properties of C. nervosum fruit extract by in vitro assays and in vitro models. C. nervosum fruit extracted using three different solvents (hexane, ethanol, and distilled water) were tested for α-amylase and α-glucosidase inhibitory activities, followed by glucose uptake in HepG2 and L6 myoblasts. Lipid accumulation in 3T3-L1 cells treated with C. nervosum fruit extracts was then examined. The results revealed that ethanolic extract of C. nervosum fruit showed better inhibition against α-amylase (IC50 of 0.42 µg/mL) and α-glucosidase (IC50 of 0.23 µg/mL) compared with other extracts. Furthermore, ethanolic extract showed higher glucose uptake potential than the standard antidiabetic drug, metformin, in HepG2 cells. The ethanolic extracts resulted in enhanced glucose utilization in L6 myoblasts compared to untreated control. All extractions showed no significantly increased lipid accumulation in 3T3-L1 cells compared to the untreated control cells. The investigation confirmed that the ethanolic extract exhibited the highest antidiabetic activity among all extracts. These results imply that C. nervosum fruit extract has antidiabetic properties and therefore they may be used as useful therapeutic agents for treating diabetes.

Anti-HIV-1 reverse transcriptase property of some edible mushrooms in Asia.

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS), which is a serious health threat worldwide. One of its core enzymes, reverse transcriptase (RT), is a target for HIV inhibition. A number of bioactive compounds have been successfully used for HIV treatment. However, HIV rapidly mutates, and long-term treatment can cause drug-resistant strains. Therefore, new inhibitors are required to overcome this problem. In this study, the aqueous, ethanolic and hexane crude extracts of 19 edible and medicinal mushrooms, which are widely grown and available commercially in Thailand, were screened against HIV-1 RT. The results showed that the water extracts of A. blazei and I. obliquus, the ethanol extracts of I. obliquus and P. igniarius and the hexane extract of I. obliquus exhibited strong anti-HIV-1 RT activity with IC50 values of 1.92 ± 0.15, 4.39 ± 0.79, 6.17 ± 0.76 and 7.75 ± 246 µg/ml, respectively. These mushrooms have the potential for HIV treatment, and further study on identification of the bioactive compounds against HIV-1 RT should be performed.

Characterization of New DNA Aptamers for Anti-HIV-1 Reverse Transcriptase.

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC50 value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.

Anti-feline immunodeficiency virus reverse transcriptase properties of some medicinal and edible mushrooms.

BACKGROUND AND AIM: Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in domestic and wild cats. Treatment of infected cats has been performed using human anti-HIV drugs, which showed some limitations. This study aimed to determine the anti-FIV potential of some mushrooms. MATERIALS AND METHODS: A total of 17 medicinal and edible mushrooms were screened to find their inhibitory effect against FIV reverse transcriptase (FIV-RT). Three solvents, water, ethanol, and hexane, were used to prepare crude mushroom extracts. Fluorescence spectroscopy was used to perform relative inhibition and 50% inhibitory concentrations (IC50) studies. RESULTS: The ethanol extract from dried fruiting bodies of Inonotus obliquus showed the strongest inhibition with an IC50 value of 0.80±0.16 µg/mL. The hexane extract from dried mycelium of I. obliquus and ethanol and water extracts from fresh fruit bodies of Phellinus igniarius also exhibited strong activities with the IC50 values of 1.22±0.20, 4.33±0.39, and 6.24±1.42 µg/mL, respectively. The ethanol extract from fresh fruiting bodies of Cordyceps sinensis, hexane extracts from dried mycelium of I. obliquus, ethanol extracts of Ganoderma lucidum, hexane extracts of fresh fruiting bodies of Morchella esculenta, and fresh fruiting bodies of C. sinensis showed moderate anti-FIV-RT activities with IC50 values of 29.73±12.39, 49.97±11.86, 65.37±14.14, 77.59±8.31, and 81.41±17.10 µg/mL, respectively. These mushroom extracts show anti-FIV potential. CONCLUSION: The extracts from I. obliquus, P. igniarius, C. sinensis, and M. esculenta showed potential anti-FIV activity.

Acquisition of naturally acquired antibody response to Plasmodium falciparum erythrocyte membrane protein 1-DBLα and differential regulation of IgG subclasses in severe and uncomplicated malaria

Objectives:To explore whether individuals infected with Plasmodium falciparum (P.falciparum) develop antibodies directed against PfEMP1-DBLα,and to assess their IgG subclass distribution in severe and uncomplicated malaria.Methods:The anti-PfDBLα IgG and their IgG subclass distributions in plasma of severe (SM) and uncomplicated malaria (UCM) were assessed by enzyme-linked immunoabsorbent assay.The antibody profiles to P.falciparum blood stage antigens were evaluated.CD36 binding ability was determined by static receptor-binding assays.Rosette formation was performed by staining with acridine orange.Results:Significantly higher number of UCM (86.48％) than SM (57.78％) plasma contained total acquisition of specific IgG to P.falciparum antigens (P =0.000).Similar manners were seen in response to P.falciparum DBLα with significant difference (UCM,59.46％ vs SM,40.00％;P =0.014).Anti-PfDBLα-IgG1 and-IgG3 were the predominant subclasses.Similar percentage of UCM (31.82％) and SM (33.33％) plasma contained only IgG1,while 13.64％ of UCM and 27.78％ of SM plasma contained only IgG3.AntiPfTDBLα-IgG1 coexpressed with more than one subclass was noted (UCM,27.27％;SM,16.67％).Obviously,IgG1 coexpressed with IgG3 (9.09％) was observed in only UCM plasma.IgG1 was coexpressed with IgG2 in UCM (9.09％) and SM (11.11％) plasma,while IgG1 was coexpressed with IgG4 only in UCM plasma (4.55％).IgG subclasses to P.falciparum antigens were distributed in a similar manner.Only the levels of IgG1,but not IgG3 were significantly higher in UCM than in SM.Conclusions:These data suggest that individuals infected with P.falciparum can develop the anti-PfEMP1 antibodies with the major contribution of specific IgG subclasses.The balance and the levels of anti-PfDBLα IgG subclasses play a crucial role in antibody mediated protection against severe malaria.

Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-pyrimidine-based kinase inhibitors.

A series of 2,4 diamino-pyrimidines have been identified from an analysis of open access high throughput anti-malarial screening data reported by GlaxoSmithKline at the 3D7 and resistant Dd2 strains. SAR expansion has been performed using structural knowledge of the most plausible parasite target. Seventeen new analogs have been synthesized and tested against the resistant K1 strain of Plasmodium falciparum (Pf). The cytotoxicity of the compounds was assessed in Vero and A549 cells and their selectivity towards human kinases including JAK2 and EGFR were undertaken. We identified compound 5n and 5m as sub-micromolar inhibitors, with equivalent anti-malarial activity to Chloroquine (CQ). Compounds 5d and 5k, µM inhibitors of Pf, displayed improved cytotoxicity with weak inhibition of the human kinases.

Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.