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  1. Trailing TRAIL Resistance in Human Breast Adenocarcinoma Cells with Trichostatin A and Zebularine
    Wong SHM, Fang CM, Loh HS, Ngai SC
    Anticancer Agents Med Chem, 2023;23(7):817-831.
    PMID: 36380402 DOI: 10.2174/1871520623666221114095733
    AIMS: The aim of this study was to sensitize the resistant breast adenocarcinoma cells towards Tumour Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced apoptosis.

    BACKGROUND: Breast cancer is a heterogeneous disease involving complex mechanisms. TRAIL is a potential anticancer candidate for targeted treatment due to its selective killing effects on neoplastic cells. Nonetheless, resistance occurs in many cancers either intrinsically or after multiple treatments.

    OBJECTIVE: Therefore, this research investigated whether the combination of Trichostatin A (TSA) and Zebularine (Zeb) (TZ) followed by TRAIL (TZT) could sensitize the human breast adenocarcinoma cells towards apoptosis.

    METHODS: The breast adenocarcinoma cells, MDA-MB-231, MCF-7 and E-MDA-MB-231 (E-cadherin re-expressed MDA-MB-231) were treated with TSA, Zeb, TZ, TRAIL and TZT. The cells were subjected to hematoxylin and eosin (H & E) staining and FITC-Annexin V/Propidium Iodide apoptosis detection prior to proteome profiling.

    RESULTS: Based on morphological observation, apoptosis was induced in all cells treated with all treatment regimens though it was more evident for the TZT-treated cells. In the apoptosis detection analysis, TZ increased early apoptosis significantly in MDA-MB-231 and MCF-7 while TRAIL induced late apoptosis significantly in E-MDA-MB-231. Based on the proteome profiling on MDA-MB-231, TRAIL R2 and Fas expression was increased. For E-MDA-MB- 231, down-regulation of catalase, paraoxonase-2 (PON2), clusterin, an inhibitor of apoptosis proteins (IAPs) and cell stress proteins validated the notion that E-cadherin re-expression enhances TZT anti-cancer efficacy. Similar trend was observed in MCF-7 whereby TZT treatment down-regulated the anti-apoptotic catalase and PON2, increased the proapoptotic, B cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad) and Bcl-2-associated X (Bax), second mitochondria-derived activator of caspase (SMAC) and HtrA serine peptidase 2 (HTRA2) as well as TRAIL receptors (TRAIL R1 and TRAIL R2).

    CONCLUSION: TZ treatment serves as an efficient treatment regimen for MDA-MB-231 and MCF-7, while TRAIL serves as a better treatment option for E-MDA-MB-231. Therefore, future studies on E-cadherin's positive regulatory role in TRAIL-induced apoptosis are warranted.

    Matched MeSH terms: Proto-Oncogene Proteins c-bcl-2/metabolism; Inhibitor of Apoptosis Proteins
  2. Polymorphism in the pvcrt-o and pvmdr-1 genes of Plasmodium vivax associated with a better prognosis for malaria
    Alves-Junior ER, Dombroski TCD, Nakazato L, Dutra V, Neves-Costa JD, Katsuragawa TH, et al.
    Trop Biomed, 2022 Sep 01;39(3):421-427.
    PMID: 36214439 DOI: 10.47665/tb.39.3.012
    The early molecular identification of strains of Plasmodium vivax that have a worse prognosis is important to stratify the risk of complications and choice of conduct made by medical teams. Thus, the aim of the present study was to associate the presence of polymorphisms in the pvmdr-1 and pvcrt-o resistance genes of P. vivax in patients with better or worse prognosis. This cross-sectional epidemiological study was conducted based on data obtained from the records of 120 patients diagnosed with malaria in the Brazilian Amazon. The T958M and F1076L mutations of the pvmdr-1 gene had a frequency of 3.3 and 4.2%, respectively, and primo-infected patients had a 17 times greater chance of being infected with protozoa with the T958M mutation compared to patients with previous episodes. Regarding pvcrt-o, the C393T and T786C polymorphisms had a frequency of 14.2 and 3.3%, respectively, and self-declared white patients had a 3.1 times greater chance of being infected with protozoa with the C393T polymorphism. In addition, patients with this pvcrt-o polymorphism had lower concentrations of C-reactive protein, indicating a better prognosis. These data present clues of genetic indicators useful for assessing the virulence of the parasite and the prognosis of patients with vivax malaria.
    Matched MeSH terms: Protozoan Proteins/genetics; Protozoan Proteins/metabolism
  3. Fulltext Knockdown of Annexin A1 induces apoptosis, causing G2/M arrest and facilitating phagocytosis activity in human leukemia cell lines
    Hasan M, Kumolosasi E, Jantan I, Jasamai M, Nazarudin N
    Acta Pharm, 2022 Mar 01;72(1):109-122.
    PMID: 36651527 DOI: 10.2478/acph-2022-0005
    Annexin A1 (ANXA1) is an endogenous protein involved in the control of proliferation, cell cycle, phagocytosis, and apoptosis in several types of cancer. To investigate the effects of ANXA1 knockdown in leukemia cells, transfection with specific ANXA1 siRNA was performed. Cell cycle and apoptosis were analyzed using flow cytometry and a mechanism involving caspases and Bcl-2 was quantified using Western blotting. Phagocytosis activity was evaluated using hematoxylin & eosin staining. The ANXA1 expression was significantly downregulated after the knockdown and apoptosis was induced in tested cells. The expression of caspase-9 and -3 increased in U937 and Jurkat cells respectively. Bcl-2 expression was downregulated in K562 and Jurkat cells while upregulated in U937. The number of leukemic cells arrested at the G2/M phase and the phagocytosis index were significantly increased in transfected cells. This suggests that ANXA1 knockdown might be a potential approach in the therapeutic strategy for leukemia.
    Matched MeSH terms: Proto-Oncogene Proteins c-bcl-2/metabolism; Proto-Oncogene Proteins c-bcl-2/pharmacology
  4. Fulltext Evaluating the sensitivity of a commercial dengue NS1 antigen-capture ELISA for early diagnosis of acute dengue virus infection
    Kumarasamy V, Chua SK, Hassan Z, Wahab AH, Chem YK, Mohamad M, et al.
    Singapore Med J, 2007 Jul;48(7):669-73.
    PMID: 17609831
    INTRODUCTION: The aim of this report is to establish an accurate diagnosis of acute dengue virus infection early, in order to provide timely information for the management of patients and early public health control of dengue outbreak.
    METHODS: 224 serum samples from patients with a clinical diagnosis of acute dengue infection, which were subsequently confirmed by laboratory tests, were used to evaluate the performance of a commercially-available dengue NS1 antigen-capture ELISA kit.
    RESULTS: The dengue NS1 antigen-capture ELISA gave an overall sensitivity rate of 93.3 percent (209/224). The sensitivity rate was significantly higher in acute primary dengue (97.4 percent) than in acute secondary dengue (68.8 percent). In comparison, the virus isolation gave an overall positive isolation rate of 64.7 percent, with a positive rate of 70.8 percent and 28.1 percent, for acute primary dengue and acute secondary dengue, respectively. Molecular detection of dengue RNA by RT-PCR gave an overall positive detection rate of 63.4 percent, with a positive rate of 62.5 percent and 68.8 percent, for acute primary dengue and acute secondary dengue, respectively. Of the 224 acute serum samples from patients with laboratory-confirmed acute dengue infection, dengue IgM was detected in 88 specimens, comprising 68 acute primary dengue specimens and 20 acute secondary dengue specimens. NS1 antigen-capture ELISA kit gave an overall sensitivity rate of 88.6 percent in the presence of anti-dengue IgM and 96.3 percent in the absence of anti-dengue IgM.
    CONCLUSION: Of the 224 acute serum samples, the sample ages of 166 acute serum samples are known. The positive detection rate of dengue NS1 antigen-capture ELISA, on the whole, was higher than the other three established diagnostic test methods for laboratory diagnosis of acute dengue infection.
    Matched MeSH terms: Viral Nonstructural Proteins/blood*; Viral Nonstructural Proteins/immunology
  5. Fulltext No Association between the Plasmodium vivax crt-o MS334 or In9pvcrt Polymorphisms and Chloroquine Failure in a Pre-Elimination Clinical Cohort from Malaysia with a Large Clonal Expansion
    Rumaseb A, Moraes Barros RR, Sá JM, Juliano JJ, William T, Braima KA, et al.
    Antimicrob Agents Chemother, 2023 Jul 18;67(7):e0161022.
    PMID: 37314336 DOI: 10.1128/aac.01610-22
    Increasing reports of resistance to a frontline malaria blood-stage treatment, chloroquine (CQ), raises concerns for the elimination of Plasmodium vivax. The absence of an effective molecular marker of CQ resistance in P. vivax greatly constrains surveillance of this emerging threat. A recent genetic cross between CQ sensitive (CQS) and CQ resistant (CQR) NIH-1993 strains of P. vivax linked a moderate CQR phenotype with two candidate markers in P. vivax CQ resistance transporter gene (pvcrt-o): MS334 and In9pvcrt. Longer TGAAGH motif lengths at MS334 were associated with CQ resistance, as were shorter motifs at the In9pvcrt locus. In this study, high-grade CQR clinical isolates of P. vivax from a low endemic setting in Malaysia were used to investigate the association between the MS334 and In9pvcrt variants and treatment efficacy. Among a total of 49 independent monoclonal P. vivax isolates assessed, high-quality MS334 and In9pvcrt sequences could be derived from 30 (61%) and 23 (47%), respectively. Five MS334 and six In9pvcrt alleles were observed, with allele frequencies ranging from 2 to 76% and 3 to 71%, respectively. None of the clinical isolates had the same variant as the NIH-1993 CQR strain, and none of the variants were associated with CQ treatment failure (all P > 0.05). Multi-locus genotypes (MLGs) at 9 neutral microsatellites revealed a predominant P. vivax strain (MLG6) accounting for 52% of Day 0 infections. The MLG6 strain comprised equal proportions of CQS and CQR infections. Our study reveals complexity in the genetic basis of CQ resistance in the Malaysian P. vivax pre-elimination setting and suggests that the proposed pvcrt-o MS334 and In9pvcrt markers are not reliable markers of CQ treatment efficacy in this setting. Further studies are needed in other endemic settings, applying hypothesis-free genome-wide approaches, and functional approaches to understand the biological impact of the TGAAGH repeats linked to CQ response in a cross are warranted to comprehend and track CQR P. vivax.
    Matched MeSH terms: Protozoan Proteins/genetics; Protozoan Proteins/therapeutic use
  6. Fulltext Effect of Secretion Efficiency of Mutant KRAS Neoantigen by Lactococcus lactis on the Immune Response of a Mucosal Vaccine Delivery Vehicle Targeting Colorectal Cancer
    Alias NAR, Hoo WPY, Siak PY, Othman SS, Mohammed Alitheen NB, In LLA, et al.
    Int J Mol Sci, 2023 May 18;24(10).
    PMID: 37240273 DOI: 10.3390/ijms24108928
    Colorectal cancer (CRC) is often caused by mutations in the KRAS oncogene, making KRAS neoantigens a promising vaccine candidate for immunotherapy. Secreting KRAS antigens using live Generally Recognized as Safe (GRAS) vaccine delivery hosts such as Lactococcus lactis is deemed to be an effective strategy in inducing specific desired responses. Recently, through the engineering of a novel signal peptide SPK1 from Pediococcus pentosaceus, an optimized secretion system was developed in the L. lactis NZ9000 host. In this study, the potential of the L. lactis NZ9000 as a vaccine delivery host for the production of two KRAS oncopeptides (mutant 68V-DT and wild-type KRAS) through the use of the signal peptide SPK1 and its mutated derivative (SPKM19) was investigated. The expression and secretion efficiency analyses of KRAS peptides from L. lactis were performed in vitro and in vivo in BALB/c mice. Contradictory to our previous study using the reporter staphylococcal nuclease (NUC), the yield of secreted KRAS antigens mediated by the target mutant signal peptide SPKM19 was significantly lower (by ~1.3-folds) compared to the wild-type SPK1. Consistently, a superior elevation of IgA response against KRAS aided by SPK1 rather than mutant SPKM19 was observed. Despite the lower specific IgA response for SPKM19, a positive IgA immune response from mice intestinal washes was successfully triggered following immunization. Size and secondary conformation of the mature proteins are suggested to be the contributing factors for these discrepancies. This study proves the potential of L. lactis NZ9000 as a host for oral vaccine delivery due to its ability to evoke the desired mucosal immune response in the gastrointestinal tract of mice.
  7. Fulltext Genetic and Global Epigenetic Modification, Which Determines the Phenotype of Transgenic Rice?
    Fan X, Chen J, Wu Y, Teo C, Xu G, Fan X
    Int J Mol Sci, 2020 Mar 06;21(5).
    PMID: 32155767 DOI: 10.3390/ijms21051819
    Transgenic technologies have been applied to a wide range of biological research. However, information on the potential epigenetic effects of transgenic technology is still lacking. Here, we show that the transgenic process can simultaneously induce both genetic and epigenetic changes in rice. We analyzed genetic, epigenetic, and phenotypic changes in plants subjected to tissue culture regeneration, using transgenic lines expressing the same coding sequence from two different promoters in transgenic lines of two rice cultivars: Wuyunjing7 (WYJ7) and Nipponbare (NP). We determined the expression of OsNAR2.1 in two overexpression lines generated from the two cultivars, and in the RNA interference (RNAi) OsNAR2.1 line in NP. DNA methylation analyses were performed on wild-type cultivars (WYJ7 and NP), regenerated lines (CK, T0 plants), segregation-derived wild-type from pOsNAR2.1-OsNAR2.1 (SDWT), pOsNAR2.1-OsNAR2.1, pUbi-OsNAR2.1, and RNAi lines. Interestingly, we observed global methylation decreased in the T0 regenerated line of WYJ7 (CK-WJY7) and pOsNAR2.1-OsNAR2.1 lines but increased in pUbi-OsNAR2.1 and RNAi lines of NP. Furthermore, the methylation pattern in SDWT returned to the WYJ7 level after four generations. Phenotypic changes were detected in all the generated lines except for SDWT. Global methylation was found to decrease by 13% in pOsNAR2.1-OsNAR2.1 with an increase in plant height of 4.69% compared with WYJ7, and increased by 18% in pUbi-OsNAR2.1 with an increase of 17.36% in plant height compared with NP. This suggests an absence of a necessary link between global methylation and the phenotype of transgenic plants with OsNAR2.1 gene over-expression. However, epigenetic changes can influence phenotype during tissue culture, as seen in the massive methylation in CK-WYJ7, T0 regenerated lines, resulting in decreased plant height compared with the wild-type, in the absence of a transformed gene. We conclude that in the transgenic lines the phenotype is mainly determined by the nature and function of the transgene after four generations of transformation, while the global epigenetic modification is dependent on the genetic background. Our research suggests an innovative insight in explaining the reason behind the occurrence of transgenic plants with random and undesirable phenotypes.
    Matched MeSH terms: Plant Proteins/genetics; Plant Proteins/metabolism*
  8. Fulltext Mutated HRAS Activates YAP1-AXL Signaling to Drive Metastasis of Head and Neck Cancer
    Jagadeeshan S, Prasad M, Badarni M, Ben-Lulu T, Liju VB, Mathukkada S, et al.
    Cancer Res, 2023 Apr 04;83(7):1031-1047.
    PMID: 36753744 DOI: 10.1158/0008-5472.CAN-22-2586
    The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression.

    SIGNIFICANCE: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.

  9. Fulltext Inhibition of SARS-CoV-2 3CL protease by the anti-viral chimeric protein RetroMAD1
    Chan LC, Mat Yassim AS, Ahmad Fuaad AAH, Leow TC, Sabri S, Radin Yahaya RS, et al.
    Sci Rep, 2023 Nov 17;13(1):20178.
    PMID: 37978223 DOI: 10.1038/s41598-023-47511-z
    COVID-19 results from SARS-CoV-2, which mutates frequently, challenging current treatments. Therefore, it is critical to develop new therapeutic drugs against this disease. This study explores the interaction between SARS-CoV-2 3CLpro and RetroMAD1, a well-characterized coronavirus protein and potential drug target, using in-silico methods. The analysis through the HDOCK server showed stable complex formation with a binding energy of -12.3, the lowest among reference drugs. The RetroMAD1-3CLpro complex underwent a 100 ns molecular dynamics simulation (MDS) in an explicit solvation system, generating various trajectories, including RMSD, RMSF, hydrogen bonding, radius of gyration, and ligand binding energy. MDS results confirmed intact interactions within the RetroMAD1-3CLpro complex during simulations. In vitro experiments validated RetroMAD1's ability to inhibit 3CLpro enzyme activity and prevent SARS-CoV-2 infection in human bronchial cells. RetroMAD1 exhibited antiviral efficacy comparable to Remdesivir without cytotoxicity at effective concentrations. These results suggest RetroMAD1 as a potential drug candidate against SARS-CoV-2, warranting further in vivo and clinical studies to assess its efficiency.
    Matched MeSH terms: Recombinant Fusion Proteins; Viral Nonstructural Proteins/metabolism
  10. MicroRNA regulation of CTP synthase and cytoophidium in Drosophila melanogaster
    Dzaki N, Woo WK, Thangadurai S, Azzam G
    Exp Cell Res, 2019 12 15;385(2):111688.
    PMID: 31678212 DOI: 10.1016/j.yexcr.2019.111688
    CTPsyn is a crucial metabolic enzyme which synthesizes CTP nucleotides. It has the extraordinary ability to compartmentalize into filaments termed cytoophidia. Though the structure is evolutionarily conserved across kingdoms, the mechanisms behind their formation remain unknown. MicroRNAs (miRNAs) are short single-stranded RNA capable of directing mRNA silencing and degradation. D. melanogaster has a high total gene count to miRNA gene number ratio, alluding to the possibility that CTPsyn too may come under their regulation. A thorough miRNA overexpression involving 123 miRNAs was conducted, followed by CTPsyn-specific staining upon cytoophidia-rich egg chambers. This revealed a small group of candidates which confer either a lengthening or truncating effect on cytoophidia, suggesting they may play a role in regulating CTPsyn. MiR-975 and miR-1014 are both cytoophidia-elongating, whereas miR-190 and miR-932 are cytoophidia-shortening. Though target prediction shows that miR-975 and miR-932 do indeed have binding sites on CTPsyn mRNA, in vitro assays instead revealed a low probability of this being true, instead indicating that the effects asserted by overexpressed miRNAs indirectly reach CTPsyn and its cytoophidia through the actions of middling elements. In silico target prediction and qPCR quantification indicated that, at least for miR-932 and miR-1014, these undetermined elements may be players in fat metabolism. This is the first study to thoroughly investigate miRNAs in connection to CTPsyn expression and activity in any species. The findings presented could serve as a basis for further queries into not only the fundamental aspects of the enzyme's regulation, but may uncover new facets of closely related pathways as well.
    Matched MeSH terms: Drosophila Proteins/genetics; Drosophila Proteins/metabolism*
  11. Fulltext The structure and dynamics of BmR1 protein from Brugia malayi: in silico approaches
    Khor BY, Tye GJ, Lim TS, Noordin R, Choong YS
    Int J Mol Sci, 2014 Jun 19;15(6):11082-99.
    PMID: 24950179 DOI: 10.3390/ijms150611082
    Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in "Brugia Rapid". However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.
    Matched MeSH terms: Helminth Proteins/metabolism; Helminth Proteins/chemistry*
  12. High Phenotypic Variability among Representative Strains of Common Salmonella enterica Serovars with Possible Implications for Food Safety
    Abdullah WZW, Mackey BM, Karatzas KAG
    J Food Prot, 2018 Jan;81(1):93-104.
    PMID: 29271685 DOI: 10.4315/0362-028X.JFP-17-190
    Salmonella is an important foodborne pathogen, whose ability to resist stress and survive can vary among strains. This variability is normally not taken into account when predictions are made about survival in foods with negative consequences. Therefore, we examined the contribution of variable phenotypic properties to survival under stress in 10 Salmonella serovars. One strain (Typhimurium 10) was intentionally RpoS-negative; however, another strain (Heidelberg) showed an rpoS mutation, rendering it inactive. We assessed an array of characteristics (motility, biofilm formation, bile resistance, acid resistance, and colony morphology) that show major variability among strains associated with a 10- to 19-fold difference between the highest and the lowest strain for most characteristics. The RpoS status of isolates did not affect variability in the characteristics, with the exception of resistance to NaCl, acetic acid, lactic acid, and the combination of acetic acid and salt, where the variability between the highest and the lowest strain was reduced to 3.1-fold, 1.7-fold, 2-fold, and 1.7-fold, respectively, showing that variability was significant among RpoS-positive strains. Furthermore, we also found a good correlation between acid resistance and lysine decarboxylase activity, showing its importance for acid resistance, and demonstrated a possible role of RpoS in the lysine decarboxylase activity in Salmonella.
    Matched MeSH terms: Bacterial Proteins/genetics*; Bacterial Proteins/chemistry
  13. Purification of recombinant antigen BmSXP used in panLF rapid kit for lymphatic filariasis
    Khoo TK, Noordin R, Santhanam A
    Indian J Exp Biol, 2012 Apr;50(4):256-64.
    PMID: 22611913
    A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.
    Matched MeSH terms: Recombinant Proteins/analysis; Recombinant Proteins/isolation & purification
  14. How the immune mousetrap works: Structural evidence for the immunomodulatory action of a peptide from influenza NS1 protein
    Zabrodskaya Y, Tsvetkov V, Shurygina AP, Vasyliev K, Shaldzhyan A, Gorshkov A, et al.
    Biophys Chem, 2024 Apr;307:107176.
    PMID: 38219420 DOI: 10.1016/j.bpc.2024.107176
    One of the critical stages of the T-cell immune response is the dimerization of the intramembrane domains of T-cell receptors (TCR). Structural similarities between the immunosuppressive domains of viral proteins and the transmembrane domains of TCR have led several authors to hypothesize the mechanism of immune response suppression by highly pathogenic viruses: viral proteins embed themselves in the membrane and act on the intramembrane domain of the TCRalpha subunit, hindering its functional oligomerization. It has also been suggested that this mechanism is used by influenza A virus in NS1-mediated immunosuppression. We have shown that the peptide corresponding to the primary structure of the potential immunosuppressive domain of NS1 protein (G51) can reduce concanavalin A-induced proliferation of PBMC cells, as well as in vitro, G51 can affect the oligomerization of the core peptide corresponding to the intramembrane domain of TCR, using AFM and small-angle neutron scattering. The results obtained using in cellulo and in vitro model systems suggest the presence of functional interaction between the NS1 fragment and the intramembrane domain of the TCR alpha subunit. We have proposed a possible scheme for such interaction obtained by computer modeling. This suggests the existence of another NS1-mediated mechanism of immunosuppression in influenza.
    Matched MeSH terms: Viral Proteins; Viral Nonstructural Proteins/chemistry
  15. Fulltext Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB)
    Usman MG, Rafii MY, Martini MY, Yusuff OA, Ismail MR, Miah G
    Cell Stress Chaperones, 2018 Mar;23(2):223-234.
    PMID: 28812232 DOI: 10.1007/s12192-017-0836-3
    Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC1F1, BC2F1 and BC3F1. The average recipient allele of the selected four BC1F1 plants was 80.75% which were used to produce the BC2F1 generation. BC1-P7 was the best BC1F1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC3F1 was 95.37%. Hsp gene expression analysis was carried out on BC1F1, BC2F1 and BC3F1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.
    Matched MeSH terms: HSP70 Heat-Shock Proteins/genetics*; HSP70 Heat-Shock Proteins/metabolism
  16. Fulltext Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis
    Faizal AM, Elias MH, Jin NM, Abu MA, Syafruddin SE, Zainuddin AA, et al.
    Front Endocrinol (Lausanne), 2024;15:1274376.
    PMID: 38524634 DOI: 10.3389/fendo.2024.1274376
    The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.
    Matched MeSH terms: Intercellular Signaling Peptides and Proteins/genetics; Intercellular Signaling Peptides and Proteins/metabolism
  17. Fulltext Characterization of Plaque Variants and the Involvement of Quasi-Species in a Population of EV-A71
    Mandary MB, Masomian M, Ong SK, Poh CL
    Viruses, 2020 Jun 17;12(6).
    PMID: 32560288 DOI: 10.3390/v12060651
    Viral plaque morphologies in human cell lines are markers for growth capability and they have been used to assess the viral fitness and selection of attenuated mutants for live-attenuated vaccine development. In this study, we investigate whether the naturally occurring plaque size variation reflects the virulence of the variants of EV-A71. Variants of two different plaque sizes (big and small) from EV-A71 sub-genotype B4 strain 41 were characterized. The plaque variants displayed different in vitro growth kinetics compared to the parental wild type. The plaque variants showed specific mutations being present in each variant strain. The big plaque variants showed four mutations I97L, N104S, S246P and N282D in the VP1 while the small plaque variants showed I97T, N237T and T292A in the VP1. No other mutations were detected in the whole genome of the two variants. The variants showed stable homogenous small plaques and big plaques, respectively, when re-infected in rhabdomyosarcoma (RD) and Vero cells. The parental strain showed faster growth kinetics and had higher viral RNA copy number than both the big and small plaque variants. Homology modelling shows that both plaque variants have differences in the structure of the VP1 protein due to the presence of unique spontaneous mutations found in each plaque variant This study suggests that the EV-A71 sub-genotype B4 strain 41 has at least two variants with different plaque morphologies. These differences were likely due to the presence of spontaneous mutations that are unique to each of the plaque variants. The ability to maintain the respective plaque morphology upon passaging indicates the presence of quasi-species in the parental population.
    Matched MeSH terms: Capsid Proteins/genetics; Capsid Proteins/metabolism
  18. Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan
    Kawalek MD, Benjamin S, Lee HL, Gill SS
    Appl Environ Microbiol, 1995 Aug;61(8):2965-9.
    PMID: 7487029
    A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
    Matched MeSH terms: Bacterial Proteins/isolation & purification*; Hemolysin Proteins
  19. Fulltext Nanotechnology against the novel coronavirus (severe acute respiratory syndrome coronavirus 2): diagnosis, treatment, therapy and future perspectives
    Rashidzadeh H, Danafar H, Rahimi H, Mozafari F, Salehiabar M, Rahmati MA, et al.
    Nanomedicine (Lond), 2021 Mar;16(6):497-516.
    PMID: 33683164 DOI: 10.2217/nnm-2020-0441
    COVID-19, as an emerging infectious disease, has caused significant mortality and morbidity along with socioeconomic impact. No effective treatment or vaccine has been approved yet for this pandemic disease. Cutting-edge tools, especially nanotechnology, should be strongly considered to tackle this virus. This review aims to propose several strategies to design and fabricate effective diagnostic and therapeutic agents against COVID-19 by the aid of nanotechnology. Polymeric, inorganic self-assembling materials and peptide-based nanoparticles are promising tools for battling COVID-19 as well as its rapid diagnosis. This review summarizes all of the exciting advances nanomaterials are making toward COVID-19 prevention, diagnosis and therapy.
    Matched MeSH terms: Proteins/therapeutic use; Proteins/chemistry
  20. Fulltext Physiological and biochemical responses of Kinnow mandarin grafted on diploid and tetraploid Volkamer lemon rootstocks under different water-deficit regimes
    Khalid MF, Hussain S, Anjum MA, Morillon R, Ahmad S, Ejaz S, et al.
    PLoS One, 2021;16(4):e0247558.
    PMID: 33831006 DOI: 10.1371/journal.pone.0247558
    Water shortage is among the major abiotic stresses that restrict growth and productivity of citrus. The existing literature indicates that tetraploid rootstocks had better water-deficit tolerance than corresponding diploids. However, the associated tolerance mechanisms such as antioxidant defence and nutrient uptake are less explored. Therefore, we evaluated physiological and biochemical responses (antioxidant defence, osmotic adjustments and nutrient uptake) of diploid (2x) and tetraploid (4x) volkamer lemon (VM) rootstocks grafted with kinnow mandarin (KM) under two water-deficit regimes. The KM/4xVM (VM4) and KM/2xVM (VM2) observed decrease in photosynthetic variables, i.e., photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), leaf greenness (SPAD), dark adopted chlorophyll fluorescence (Fv/Fm), dark adopted chlorophyll fluorescence (Fv´/Fm´), relative water contents (RWC) and leaf surface area (LSA), and increase in non-photochemical quenching (NPQ) under both water-deficit regimes. Moreover, oxidative stress indicators, i.e., malondialdehyde (MDA) and hydrogen peroxide, and activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APx), glutathione reductase (GR) were increased under both water-deficit regimes. Nonetheless, increase was noted in osmoprotectants such as proline (PRO) and glycine betaine (GB) and other biochemical compounds, including antioxidant capacity (AC), total phenolic content (TPC) and total soluble protein (TSP) in VM2 and VM4 under both water-deficit regimes. Dry biomass (DB) of both rootstocks was decreased under each water-deficit condition. Interestingly, VM4 showed higher and significant increase in antioxidant enzymes, osmoprotectants and other biochemical compounds, while VM2 exhibited higher values for oxidative stress indicators. Overall, results indicated that VM4 better tolerated water-deficit stress by maintaining photosynthetic variables associated with strong antioxidant defence machinery as compared to VM2. However, nutrient uptake was not differed among tested water-deficit conditions and rootstocks. The results conclude that VM4 can better tolerate water-deficit than VM2. Therefore, VM4 can be used as rootstock in areas of high-water deficiency for better citrus productivity.
    Matched MeSH terms: Plant Proteins/genetics; Plant Proteins/metabolism*
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