Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the
overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor
(CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is
specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are
able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering
it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human
T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction
protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene.
CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted
into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These
lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabeadconjugated
CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected
and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models
to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells.
Salmonella enterica subsp. enterica serovar Stanley (S. Stanley) is a pathogen that contaminates food, and is related to Salmonella outbreaks in a variety of hosts such as humans and farm animals through products like dairy items and vegetables. Despite the fact that several vaccines of Salmonella strains had been constructed, none of them were developed according to serovar Stanley up to this day. This study presents results of genome sequencing and analysis on our S. Stanley UPM 517 strain taken from fecal swabs of 21-day-old healthy commercial chickens in Perak, Malaysia and used Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) as a reference to be compared with. First, sequencing and assembling of the Salmonella Stanley UPM 517 genome into a contiguous form were done. The work was then continued with scaffolding and gap filling. Annotation and alignment of the draft genome was performed with S. Typhimurium LT2. The other elements of virulence estimated in this study included Salmonella pathogenicity islands, resistance genes, prophages, virulence factors, plasmid regions, restriction-modification sites and the CRISPR-Cas system. The S. Stanley UPM 517 draft genome had a length of 4,736,817 bp with 4,730 coding sequence and 58 RNAs. It was discovered via genomic analysis on this strain that there were antimicrobial resistance properties toward a wide variety of antibiotics. Tcf and ste, the two fimbrial virulence clusters related with human and broiler intestinal colonizations which were not found in S. Typhimurium LT2, were atypically discovered in the S. Stanley UPM 517 genome. These clusters are involved in the intestinal colonization of human and broilers, respectively. There were seven Salmonella pathogenicity islands (SPIs) within the draft genome, which contained the virulence factors associated with Salmonella infection (except SPI-14). Five intact prophage regions, mostly comprising of the protein encoding Gifsy-1, Fels-1, RE-2010 and SEN34 prophages, were also encoded in the draft genome. Also identified were Type I-III restriction-modification sites and the CRISPR-Cas system of the Type I-E subtype. As this strain exhibited resistance toward numerous antibiotics, we distinguished several genes that had the potential for removal in the construction of a possible vaccine candidate to restrain and lessen the pervasiveness of salmonellosis and to function as an alternative to antibiotics.
In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza.
Therapeutic gene editing is becoming more feasible with the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system. However, the successful implementation of CRISPR/Cas9-based therapeutics requires a safe and efficient in vivo delivery of the CRISPR components, which remains challenging. This study presents successful preparation, optimization, and characterization of alginate nanoparticles (ALG NPs), loaded with two CRISPR plasmids, using electrospray technique. The aim of this delivery system is to edit a target gene in another plasmid (green fluorescent protein (GFP)). The effect of formulation and process variables were evaluated. CRISPR ALG NPs showed mean size and zeta potential of 228 nm and -4.42 mV, respectively. Over 99.0% encapsulation efficiency was achieved while preserving payload integrity. The presence of CRISPR plasmids in the ALG NPs was confirmed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. The tests revealed that the nanoparticles were cytocompatible and successfully introduced the Cas9 transgene in HepG2 cells. Nanoparticles-transfected HepG2 was able to edit its target plasmid by introducing double-strand break (DSB) in GFP gene, indicating the bioactivity of CRISPR plasmids encapsulated in alginate nanoparticles. This suggests that this method is suitable for biomedical application in vitro or ex vivo. Future investigation of theses nanoparticles might result in nanocarrier suitable for in vivo delivery of CRISPR/Cas9 system.
Salinivibrio proteolyticus M318, a halophilic bacterium isolated from fermented shrimp paste, is able to produce polyhydroxyalkanoate (PHA) from different carbon sources. In this study, we report the whole-genome sequence of strain M138, which comprises 2 separated chromosomes and 2 plasmids, and the complete genome contains 3,605,935 bp with an average GC content of 49.9%. The genome of strain M318 contains 3341 genes, 98 tRNA genes, and 28 rRNA genes. The 16S rRNA gene sequence and average nucleotide identity analysis associated with morphological and biochemical tests showed that this strain has high homology to the reference strain Salinivibrio proteolyticus DSM 8285. The genes encoding key enzymes for PHA and ectoine synthesis were identified from the bacterial genome. In addition, the TeaABC transporter responsible for ectoine uptake from the environment and the operon doeABXCD responsible for the degradation of ectoine were also detected. Strain M318 was able to produce poly(3-hydroxybutyrate) [P(3HB)] from different carbon sources such as glycerol, maltose, glucose, fructose, and starch. The ability to produce ectoines at different NaCl concentrations was investigated. High ectoine content of 26.2% of cell dry weight was obtained by this strain at 18% NaCl. This report provides genetic information regarding adaptive mechanisms of strain M318 to stress conditions, as well as new knowledge to facilitate the application of this strain as a bacterial cell factory for the production of PHA and ectoine.
Enterovirus 71 (EV71) is the major causative agent in hand, foot, and mouth disease (HFMD), and it mainly infects children worldwide. Despite the risk, there is no effective vaccine available for this disease. Hence, a recombinant protein construct of truncated nucleocapsid protein viral protein 1 (NPt-VP1198-297), which is capable of inducing neutralizing antibody against EV71, was evaluated in a mouse model. Truncated nucleocapsid protein Newcastle disease virus that was used as immunological carrier fused to VP1 of EV71 as antigen. The recombinant plasmid carrying corresponding genes was constructed by recombinant DNA technology and the corresponding protein was produced in Escherichia coli expression system. The recombinant NPt-VP1198-297 protein had elicited neutralizing antibodies against EV71 with the titer of 1:16, and this result is higher than the titer that is elicited by VP1 protein alone (1:8). It was shown that NPt containing immunogenic epitope(s) of VP1 was capable of inducing a greater functional immune response when compared to full-length VP1 protein alone. It was capable to carry larger polypeptide compared to full-length NP protein. The current study also proved that NPt-VP1198-297 protein can be abundantly produced in recombinant protein form by E. coli expression system. The findings from this study support the importance of neutralizing antibodies in EV71 infection and highlight the potential of the recombinant NPt-VP1198-297 protein as EV71 vaccine.
The present study aimed to screen plants for bioactive compounds with potential antibacterial activities. In our efforts to evaluate plants from Borneo, we isolated and elucidated the structures of four natural products from the bioactive fraction of a chloroform extract of Goniothalamus longistipetes using various chromatographic and spectroscopic techniques. The bioactive compounds were identified as a known styryllactone, (+)-altholactone ((2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (1), a new styryllactone, (2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (2) as well as a new alkaloid, 2,6-dimethoxyisonicotinaldehyde (3) and a new alkenyl-5-hydroxyl-phenyl benzoic acid (4). 1 and 4 showed broad-spectrum anti-bacterial activities against Gram-positive and Gram-negative bacteria as well as acid-fast model selected for this study. Compound 2 only demonstrated activities against Gram-positive bacteria whilst 3 displayed selective inhibitory activities against Gram-positive bacterial strains. Additionally, their mechanisms of anti-bacterial action were also investigated. Using Mycobacterium smegmatis as a fast-growing model of tubercle bacilli, compounds 1, 2 and 4 demonstrated inhibitory activities against whole-cell drug efflux and biofilm formation; two key intrinsic mechanisms of antibiotic resistance. Interestingly, the amphiphilic compound 4 exhibited inhibitory activity against the conjugation of plasmid pKM101 in Escherichia coli using a plate conjugation assay. Plasmid conjugation is a mechanism by which Gram-positive and Gram-negative-bacteria acquire drug resistance and virulence. These results indicated that bioactive compounds isolated from Goniothalamus longistipetes can be potential candidates as 'hits' for further optimisation.
Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.
Fifty five (n=55) isolates of Escherichia coli isolated from ducks in Penang, Malaysia were examined for their susceptibility to eleven different antibiotics and assayed for the presence of plasmid DNAs. All the 55 Escherichia coli isolates were resistant (100%) to vancomycin. Higher resistance (= 60) occurred for tetracycline 51 (92.7%), ampicillin 40 (72.7%), streptomycin 37 (67.3%), and sulfamethoxazole-trimethophrim 37 (67.3%). No and low resistance was observed for nitrofurantoin (0%) and gentamicin (1.8%), respectively. The isolates also showed some intermediate resistances to all antibiotics examined except for vancomycin. The 55 Escherichia coli isolates exhibited 23 different antibiotic resistant patterns with MAR index ranging from 0.09-0.82. Majority of the Escherichia coli isolates exhibited resistant pattern of VA-C-OFX-SXT-TE-AMP-NA-KF and VA-S-C-OFX-SXT-TE-AMP-NA-KF with MAR index of 0.73 and 0.82, respectively. The smallest plasmid DNA size was 1.2 kb and the largest plasmid DNA size was 81.5 kb. 51 (93%) of the duck Escherichia coli isolates harbored plasmids. The was no direct correlation between plasmid DNA sizes and antibiotic resistant among the duck Escherichia coli isolates. Thus, the antibiotic resistant of the Escherichia coli isolates could mostly be mediated by chromosomes instead of plasmids. This study also suggests that the use of antibiotics in duck farming in Penang, Malaysia needs to be controlled to prevent the spread of multiple antibiotic resistant Escherichia coli isolates.
Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.
Anomalies in DNA replication, repair and recombination in ataxia-telangiectasia (A-T) point to a defect in structure or function of chromatin. In this study we have compared DNA-protein binding in nuclear extracts from control and A-T cells using two assay systems, filter-binding and DNA-accessibility. Interestingly, the extent of DNA protein binding over a range of protein concentration was significantly lower in A-T extracts. In addition the accessibility of the restriction enzyme Eco R1 to protein-bound plasmid was greater when A-T extracts were used. This is in keeping with the reduced binding observed in the filter-binding assay.
One hundred and thirty eight penicillinase producing Neisseria gonorrhoeae (PPNG) and 239 non-PPNG strains were characterised serologically using a panel of seven monoclonal antibodies directed against protein 1A and seven against protein 1B. An association between serovar and susceptibility to antimicrobial agents, auxotype, and plasmid content was observed. Serogroup WI strains were more sensitive to penicillin, ampicillin, tetracycline, erythromycin, cefoxitin, and cefuroxime. Sixty five (82%) of the 79 WI strains were typed as being serovar Aedgkih, and 47 (72%) of these strains required arginine, uracil, and hypoxanthine for growth (AUH-). Seventy one (44%) of 160 WII/WIII strains were serovar Bacejk, and 42 (59%) of these required proline, citrulline, and uracil for growth (PCU-) and were plasmid free. Serovars Bcgk, Beghjk, Bacjk, and Bajk were associated with resistance to antimicrobial agents. Analysis of PPNG isolates showed a new serovar, Af, which was associated with strains imported from Malaysia and Singapore that required proline and ornithine for growth (Pro-Orn-) and carried the 24.5 megadalton transfer plasmid, the 2.6 megadalton cryptic plasmid, and the 4.5 megadalton penicillinase producing plasmid. Other associations between serovar and geographical location were noted.
Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic
acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 µM galactose and 100
µM tyrosine supplemented with 600 µM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 µM galactose and 50 µM tyrosine with 200 µM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative
T. semidecandra transformants was verified by PCR amplification with specific primers.
Vibrio parahaemolyticus is one of the most widely recognized pathogenic Vibrio species due to numerous outbreaks and its’ wide occurrence in marine environment. In this study, 32 isolates of V. parahaemolyticus isolated from cockles were tested for sensitivity to 16 antibiotics and the presence of plasmids. All the isolates were multi-resistance, defined as resistant to atleast three different antibiotics with multiple antibiotic resistance indexes ranging from 0.31 to 0.69, indicating the isolates originate from high risk sources of contamination where antibiotics are often used. In the plasmid profiling test, only 15 isolates (47%) harbored plasmid DNA, which ranged in size from 2.7 to 56.2 kb, separating the isolates into 14 plasmid profiles. Hence, food contaminated with antibiotic resistant V. parahaemolyticus could be a major threat to public health due to the distinct possibility that they can be a significant reservoir of genes encoding antibiotic resistance determinants that can be transferred intra or interspecies. As in many developing countries, raw food hygiene and antimicrobial resistance epidemiology is still in the infancy stage in the locality of the study and thus our data provide a current baseline profile of antimicrobial resistance and plasmid of V. parahaemolyticusfrom cockles in Padang, Indonesia.
Fifty-nine isolates of Salmonella enterica subsp. enterica (S. enterica) isolated from indigenous vegetables, ‘selom’ (Oenanthe stolonifera) associated with 13 different serovars were obtained from Chemistry Department of Malaysia. The isolates encompass the common serovar, Salmonella enterica subsp. enterica serovar Weltevreden (S. Weltevreden) (39%) and Salmonella enterica subsp. enterica serovar Agona (S. Agona) (8.5%). Frequencies of the other 11 Salmonella serovars were ranged from 1.7% to 5.1%. All isolates were characterized by Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR), random amplified polymorphic DNA (RAPD), plasmid profiling and antimicrobial susceptibility testing. The results demonstrated ERIC-PCR, RAPD and composite analysis of both are suitable typing methods for S. enterica by demonstrating good discriminative ability and can be utilize as a rapid approach of comparing S. enterica isolates for epidemiological investigation. From this study, ERIC-PCR is exhibited lower discriminatory power when compare with RAPD. On the other hand, plasmid profiles yielded 32 profiles with molecular size ranging from 1129 bp to 17911 bp. Thirteen antimicrobial agents were included in this study and all isolates showed 100% (59/59) resistant to erythromycin and showed Multiple Antimicrobial Resistance (MAR) indexes ranging from 0.08 to 0.68. Dendrogram generated from antimicrobial resistance profiling exhibited poor discriminatory capability at serovar level. Although poultry still remain as the common reservoir for multidrug resistant (MDR) Salmonella. The isolation of 13 Salmonella serovars from selom that showed high MDR in this study is alarming. These results supported the notion that indigenous vegetable (selom) are gaining more antimicrobial resistance and could be potential health hazards.
Escherichia coli and Escherichia coli O157 were identified from “selom” (Oenanthe stolonifera), “pegaga” (Centella asiatica), beef, chicken, lamb, buffalo, “ulam Raja” (Cosmos caudatus) and “tenggek burung” (Euodia redlevi). The bacteria were recovered using chromagenic agar. Isolated Escherichia coli and Escherichia coli 0157 were further characterized by plasmid profiling and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The virulence genes of the isolates (VT1, VT2, LT, ST, eaeA, inV) that produces pathogenic Escherichia coli and 16S rRNA gene were screened by a multiplex PCR assay. The plasmid profiling analysis showed that out of 176 isolates, only 103 isolates contained plasmids. ERIC-PCR analysis generated amplified products in the range of ~150 bp to > 1000 bp categorizing isolates into a total of 52 different profiles. Multiplex PCR showed that 20 (32.3%) of the isolates carried eaeA gene, 6 (9.7%) isolates possessed inV genes, only 1 (1.6%) have VT2 genes and 1 (1.6%) as well carried VT1 genes, 2 (3.2%) of the isolates harboured LT genes, and only 1 (1.6%) isolate possessed ST genes. There were no correlation between plasmid, ERIC-PCR and virulence genes profiles.
A total of 87 market fish samples representing five types of fish were evaluated for the presence of Aeromonas spp. Of the samples examined, 69%, 55%, 11.5% and 2.3% harbored Aeromonas spp., A. veronii biovar sobria, A. hydrophila and A. caviae, respectively. The 60 isolated Aeromonas spp. strains were further examined for hemolytic activity, resistance to antimicrobial agents and presence of plasmids. Hemolytic activity varied widely among the isolated strains. Though all the isolates demonstrated resistance to three or more of the antibiotics tested, all were susceptible to ceptazidime. Thirty-four (56.7%) of the sixty isolates harbored plasmids, with sizes ranging from 2.3 to 15.7 kb. These results indicate that hemolytic, multiple antibiotic resistant and genetically diverse aeromonads are easily recovered from fish in this region.
A total of 31 strains of Vibrio cholerae O1 (10 from outbreak cases and 7 from surface water) and non-O1 (4 from clinical and 10 from surface water sources) isolated between 1993 and 1997 were examined with respect to presence of cholera enterotoxin (CT) gene by PCR-based assays, resistance to antibiotics, plasmid profiles and random amplified polymorphic DNA (RAPD) analysis. All were resistant to 9 or more of the 17 antibiotics tested. Identical antibiotic resistance patterns of the isolates may indicate that they share a common mode of developing antibiotic resistance. Furthermore, the multiple antibiotic resistance indexing showed that all strains tested originated from high risk contamination. Plasmid profile analysis by agarose gel electrophoresis showed the presence of small plasmids in 12 (7 non-O1 and 5 O1 serotypes) with sizes ranging 1.3-4.6 MDa. The CT gene was detected in all clinical isolates but was present in only 14 (6 O1 serotype and 8 non-O1 serotype) isolates from environmental waters. The genetic relatedness of the clinical and environmental Vibrio cholerae O1 and non-O1 strains was investigated by RAPD fingerprinting with four primers. The four primers generated polymorphisms in all 31 strains of Vibrio cholerae tested, producing bands ranging from < 250 to 4500 bp. The RAPD profiles revealed a wide variability and no correlation with the source of isolation. This study provides evidence that Vibrio cholerae O1 and non-O1 have significant public health implications.
Background: Recently, chronic kidney disease (CKD)-mineral and bone disorder (MBD) has become one of common complications occurring in CKD patients. Therefore, development of a new treatment for CKD-MBD is very important in the clinic. In China, Fucoidan (FPS), a natural compound of Laminaria japonica has been frequently used to improve renal dysfunction in CKD. However, it remains elusive whether FPS can ameliorate CKD-MBD. FGF23-Klotho signaling axis is reported to be useful for regulating mineral and bone metabolic disorder in CKD-MBD. This study thereby aimed to clarify therapeutic effects of FPS in the CKD-MBD model rats and its underlying mechanisms in vivo and in vitro, compared to Calcitriol (CTR). Methods: All male rats were divided into four groups: Sham, CKD-MBD, FPS and CTR. The CKD-MBD rat models were induced by adenine administration and uninephrectomy, and received either FPS or CTR or vehicle after induction of renal injury for 21 days. The changes in parameters related to renal dysfunction and renal tubulointerstitial damage, calcium-phosphorus metabolic disorder and bone lesion were analyzed, respectively. Furthermore, at sacrifice, the kidneys and bone were isolated for histomorphometry, immunohistochemistry and Western blot. In vitro, the murine NRK-52E cells were used to investigate regulative actions of FPS or CTR on FGF23-Klotho signaling axis, ERK1/2-SGK1-NHERF-1-NaPi-2a pathway and Klotho deficiency. Results: Using the modified CKD-MBD rat model and the cultured NRK-52E cells, we indicated that FPS and CTR alleviated renal dysfunction and renal tubulointerstitial damage, improved calcium-phosphorus metabolic disorder and bone lesion, and regulated FGF23-Klotho signaling axis and ERK1/2-SGK1-NHERF-1-NaPi-2a pathway in the kidney. In addition, using the shRNA-Klotho plasmid-transfected cells, we also detected, FPS accurately activated ERK1/2-SGK1-NHERF-1-NaPi-2a pathway through Klotho loss reversal. Conclusion: In this study, we emphatically demonstrated that FPS, a natural anti-renal dysfunction drug, similar to CTR, improves renal injury-related calcium-phosphorus metabolic disorder and bone abnormality in the CKD-MBD model rats. More importantly, we firstly found that beneficial effects in vivo and in vitro of FPS on phosphorus reabsorption are closely associated with regulation of FGF23-Klotho signaling axis and ERK1/2-SGK1-NHERF-1-NaPi-2a pathway in the kidney. This study provided pharmacological evidences that FPS directly contributes to the treatment of CKD-MBD.
Genetic intervention via the delivery of functional genes such as plasmid DNA (pDNA) and short-interfering RNA (siRNA) offers a great way to treat many single or multiple genetic defects effectively, including mammary carcinoma. Delivery of naked therapeutic genes or siRNAs is, however, short-lived due to biological clearance by scavenging nucleases and circulating monocytes. Low cellular internalization of negatively-charged nucleic acids further causes low transfection or silencing activity. Development of safe and effectual gene vectors is therefore undeniably crucial to the success of nucleic acid delivery. Inorganic nanoparticles have attracted considerable attention in the recent years due to their high loading capacity and encapsulation activity. Here we introduce strontium salt-based nanoparticles, namely, strontium sulfate, strontium sulfite and strontium fluoride as new inorganic nanocarriers. Generated strontium salt particles were found to be nanosized with high affinity towards negatively-charged pDNA and siRNA. Degradation of the particles was seen with a drop in pH, suggesting their capacity to respond to pH change and undergo dissolution at endosomal pH to release the genetic materials. While the particles are relatively nontoxic towards the cells, siRNA-loaded SrF2 and SrSO3 particles exerted superior transgene expression and knockdown activity of MAPK and AKT, leading to inhibition of their phosphorylation to a distinctive extent in both MCF-7 and 4T1 cells. Strontium salt nanoparticles have thus emerged as a promising tool for applications in cancer gene therapy.