Ratree Tavichakorntrakool

Serial analyses of postmortem changes in human skeletal muscle : A case study of alterations in proteome profile, histology, electrolyte contents, water composition, and enzyme activity

Postmortem tissues are frequently used in forensic investigation, clinical studies, and biomedical research. It is well known that the shorter period from death to analyses provides the more accurate results. However, the longest postmortem interval that still provides the reliable data remains unclear. We performed serial analyses of postmortem changes in proteome profile, histology, electrolyte contents, water composition, and enzyme activity in human vastus lateralis muscle from a male cadaver (died from a motorcycle accident). This uninjured muscle was sectioned into several 1‐cm3 cubes and stored in individual closed tubes at 4 or 25°C for 0, 2, 4, 6, 12, 24 or 48 h prior to proteomic, histological, chemical and biochemical analyses. At 4°C, the 2‐DE proteome profile remained unchanged until 24 h, when some poorly focused protein spots and significant decrease in the total number of visualized spots were observed. These changes were detectable earlier (12 h) in the samples stored at 25°C. Profound vacuolization and autolysis started at 24 and 6 h for the samples stored at 4ºC and 25°C, respectively. K and Mg contents began to increase at 12 and 48 h, respectively, for both temperatures. However, the increase in Na and Ca contents began at 24 h in the samples stored at 4°C, but started earlier (12 h) in those stored at 25°C. Water content started to decline at 48 and 24 h in the samples stored at 4 and 25°C, respectively. Muscle lactate dehydrogenase activity began to be out of range at 12 h for both temperatures. These findings demonstrate that storing the samples at 4°C could delay some of the aforementioned changes, which occurred more rapidly at 25°C. Our results also suggest that muscle proteome profile, histology, electrolyte contents, water composition, and enzyme activity should be analyzed within the optimal postmortem intervals, which vary among individual analyses, to obtain the most reliable data.

Metabolic enzymes, antioxidants, and cytoskeletal proteins are significantly altered in vastus lateralis muscle of K-depleted cadaveric subjects.

Molecular mechanisms underlying myopathy caused by prolonged potassium (K) depletion remain poorly understood. In the present study, we examined proteome profile of vastus lateralis muscle obtained from cadaveric subjects who had K depletion (KD) (muscle K<80 micromol/g wet weight) compared to those who had no KD (NKD) (muscle K>or=80 micromol/g wet weight) (n=6 per group). Muscle proteins were extracted, resolved by 2-DE, and visualized with CBB-R250 stain. Spot matching and intensity analysis revealed significant changes in levels of 11 (6 increased and 5 decreased) protein spots in the KD group. Q-TOF MS and MS/MS analyses identified these altered proteins as metabolic enzymes (aldehyde dehydrogenase 1A1, uridine diphosphoglucose pyrophosphorylase, enolase 1, cytosolic malate dehydrogenase, and carbonic anhydrase III), antioxidants (peroxiredoxin-3 isoform b), cytoskeletal proteins (slow-twitch skeletal troponin I and myosin light chain 2), and others. These altered proteins are involved in many cellular functions, including bioenergetics, acid-base regulation, oxidative stress response, and muscle contractility. Validation was done by Western blot analysis, which confirmed the increased level of peroxiredoxin-3 and decreased level of troponin-I in the KD muscle. Linear regression analysis also revealed a significant negative correlation between peroxiredoxin-3 level and muscle K content (r=-0.887; p<0.001), as well as a significant positive correlation between troponin-I level and muscle K content (r=0.618; p<0.05). Our results implicate the important roles these altered proteins play in the development of KD-associated myopathy.

SCCmec IX in meticillin-resistant Staphylococcus aureus and meticillin-resistant coagulase-negative staphylococci from pigs and workers at pig farms in Khon Kaen, Thailand

Livestock-associated meticillin-resistant Staphylococcus aureus, clonal complex (CC) 398, has been reported in Europe, whereas CC9 MRSA has mostly been found in Asia. Therefore, we aimed to detect MRSA on pig farms in north-eastern Thailand. A total of 257 nasal swabs (159 samples from pigs and 98 from pig-farm workers) were collected from three pig farms in north-eastern Thailand from 2010 to 2011. MRSA isolates were confirmed for femA and mecA genes by PCR. The MICs of eight antimicrobials, namely vancomycin (VA), cefazolin (CZ), ofloxacin (OF), tetracycline (TET), erythromycin (ER), oxacillin (OX), cefoxitin (FOX) and gentamicin (GN), were tested by agar dilution method. The virulence genes for Panton-Valentine leukocidin toxin (lukSF-PV), toxic shock syndrome toxin-1 (tst) and α-haemolysin (hla) were detected by PCR. Strain typing was performed by staphylococcal cassette chromosome (SCC) mec, agr, spa and multilocus sequence typing. Four MRSA were isolated: three from workers and one from a pig. All the MRSA isolates were resistant to OX, GN, ER, TET and CZ, and they all carried hla only. Two MRSA from humans carried SCCmec II-sequence type (ST)764-agrII, whereas the two remaining MRSA (one each from a human and a pig) contained SCCmec IX-ST9-agrII. Interestingly, meticillin-resistant coagulase-negative Staphylococcus isolates carrying SCCmec IX were also obtained from five workers and three pigs. This study suggests that the SCCmec IX element is distributed among the Staphylococcus found in pigs and pig-farm workers, and pigs may be a reservoir for MRSA in the community.

Antioxidant and antibacterial properties of selected Thai weed extracts

ABSTRACT Objective To analyze antioxidant and antibacterial properties of selected weeds commonly found in Northeast Thailand including Ageratum conyzoides L., Alysicarpus vaginalis L., Commelina bengalensis L., Euphorbia hirta L., Hyptis suaveolens L., Parthenocissus quinquefolia L., and Trianthema portulacastrum L. Methods Ferric reducing antioxidant power and radical scavenging activity of the aqueous and ethanol weed extracts were determined. Phytochemical screening, total phenolic and flavonoid contents were done. Antibacterial activity against Aeromonas hydrophila , Aeromonas caviae , Edwardsiella tarda , Plesiomonas shigelloides , Ralstonia spp., Xanthomonas campestris pv. Vesicatoria , Salmonella spp. and Shigella spp. was performed by disc diffusion assay. Results The results showed that Euphorbia hirta extract had the highest total phenolic contents and was the most effective against most of the test organisms compared to the other weed extracts. Hyptis suaveolens ethanol extract weakly inhibited Ralstonia spp. and Salmonella spp. (10.42% and 9.84% inhibition, respectively). Trianthema portulacastrum ethanol extract had 20.10% inhibition against Shigella spp. Parthenocissus quinquefolia aqueous extract strongly inhibited Aeromonas caviae and Aeromonas hydrophila with 55.90% and 59.68% inhibition, respectively. Conclusions These weeds may be serving as a potential source of antibacterial agents.

Differential colony size, cell length, and cellular proteome of Escherichia coli isolated from urine vs. stone nidus of kidney stone patients

BACKGROUND Escherichia coli is associated with kidney stone disease, as a cause or an effect (secondary or recurrent urinary tract infection, UTI). Defining phenotypic or functional differences between E. coli inside stone nidus (ECS, associated with infection-induced stone) and outside the stone (i.e. from urine) (ECU, represented secondary infection) would be helpful to better understand bacterial involvement in this disease. METHODS ECS and ECU were isolated from 100 stone formers and subjected to antimicrobial susceptibility test, ERIC-PCR genotyping, determination of biofilm formation, bacterial colony size on agar plate and cell length in broth, 2-DE, nanoLC-MS/MS, protein network analysis, and pyruvate dehydrogenase (PDH) activity assay. RESULTS From 100 stone formers, 36 had positive bacterial culture, of which 5 pairs had identical antimicrobial susceptibility patterns and comparable ERIC-PCR genotypes. ECS had smaller colony size and longer cell length than ECU. 2-DE proteomic analysis revealed significantly differential levels of proteins involved in carbohydrate metabolism, stress response, and RNA/protein metabolism. Functional validation demonstrated lower PDH activity in ECS. CONCLUSIONS All these differential phenotypic and cellular proteome findings might be adaptive response of E. coli from remote infection to survive within the stone matrix that subsequently caused recurrent UTI in kidney stone patients.

Inhibitory Effects of Gallic Acid Isolated from Caesalpinia mimosoides Lamk on Cholangiocarcinoma Cell Lines and Foodborne Pathogenic Bacteria

Gallic acid was isolated from Caesalpinia mimosoides Lamk and the structure s identified based on spectroscopic analysis and comparison with authentic compound. In this study we compared the ability of natural gallic acid (nGA) and commercial gallic acid (cGA) to inhibit the proliferation of cholangiocarcinoma cell lines (M213, M214) and foodborne pathogenic bacteria (Salmonella spp. and Plesiomonas shigelloides). Both nGA and cGA had the same inhibitory effects on cell proliferation by inducing apoptosis of cholangiocarcinoma cell lines. In addition, nGA inhibited growth of foodborne pathogenic bacteria in the same manner as cGA. Our results suggest that nGA from Caesalpinia mimosoides Lamk is a potential anticancer and antibacterial compound. However, in vivo studies are needed to elucidate the specific mechanisms involved.

Phenotypic Characteristics of Vancomycin-Non-Susceptible Staphylococcus aureus

Background: Staphylococcus aureus, with reduced vancomycin susceptibility, is probably under the regulation of several genes and various express phenotypes. Objectives: This study aimed to investigate the phenotypic differences between vancomycin-susceptible S. aureus (VSSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA) isolates. Materials and Methods: A total of 130 methicillin-resistant S. aureus (MRSA) isolates were studied, including 49 VSSA, 28 hVISA, and 5 VISA isolates from blood cultures and 48 isolates (two VSSA, six hVISA, and 40 VISA) derived in vitro (laboratory-induced/sub-passaged). Their phenotypes were examined using a coagulase tube test, colony spreading on soft agar, and urease activity. The SCCmec and agr typing were performed using multiplex PCR. Results: Most of the MRSA isolates were SCCmec III-agr I (84.5%), followed by SCCmec II-agr II (11.8%). The average plasma coagulation time of vancomycin-non-susceptible isolates was longer than that of the susceptible isolates (12 vs. 2.6 hours). Four hVISA (P = 0.023) and nine VISA (P < 0.001) isolates yielded a negative coagulase test after 24-hour incubation. The percentage of VSSA isolates showing non-spreading colonies (accessory gene regulator (agr) dysfunction) was significantly lower than in the VISA group (P = 0.013), but no significant difference was found between VSSA and hVISA. The VISA group showed higher urease activity than that of the VSSA and hVISA groups (P = 0.002). Conclusions: There were diverse phenotypic changes among vancomycin-non-susceptible S. aureus isolates. This may be due to the variety of related regulatory systems. The diversity of phenotypic expression may result in its misidentification in routine laboratory checks.

Anticancer Potential of Cratoxylum formosum Subsp. Pruniflorum (Kurz.) Gogel Extracts Against Cervical Cancer Cell Lines.

BACKGROUND Most northeast Thai vegetables may play roles in human health by acting as antioxidant and anticancer agents. Recent study showed that Cratoxylum formosum subsp. pruniflorum (Kurz.) Gogel. (Teawdang) could inhibit growth of liver cancer cell lines. Cervical cancer, which has human papilloma virus as its main cause, is found at high incidence in Thailand. Due to increasing drug resistance, searches for potential anticancer compounds from natural source are required. Therefore, our purpose was to evaluate the cytotoxicity of Teawdang extracts in cervical cancer cell lines. MATERIALS AND METHODS Teawdang edible parts, purchased from Khon Kaen market during July-October 2013 was extracted with organic solvent. Phenolic profiles of crude hexane (CHE), ethyl acetate (CEE), methanol (CME) and water (CWE) extracts were performed by high performance liquid chromatographic (HPLC) techniques. Their cytotoxic effects on cervical cancer cells were investigated with HPV-non infected (C-33A) and HPV-infected (HeLa and SiHa) cell lines. RESULTS HPLC profiles showed that all crude extracts contained caffeine, ferulic acid and resveratrol. CME and CEE had high contents of gallic acid and quercetin. Catechin was found only in CWE. Cytotoxicity test showed that CEE had the lowest IC50 on HeLa (143.18±13.35 μg/mL) and SiHa cells (106.45±15.73 μg/mL). C-33A cells were inhibited by CWE (IC50 = 130.95±3.83 μg/mL). CONCLUSIONS There were several phenolic compounds in Teawdang extracts which may have cytotoxic effects on cervical cancer cell lines. Investigation of these bioactive compounds as new sources of anticancer agents is recommended.

Elongation factor Tu on Escherichia coli isolated from urine of kidney stone patients promotes calcium oxalate crystal growth and aggregation

Escherichia coli is the most common bacterium isolated from urine and stone matrix of calcium oxalate (CaOx) stone formers. Whether it has pathogenic role(s) in kidney stone formation or is only entrapped inside the stone remains unclear. We thus evaluated differences between E. coli isolated from urine of patients with kidney stone (EUK) and that from patients with urinary tract infection (UTI) without stone (EUU). From 100 stone formers and 200 UTI patients, only four pairs of EUK/EUU isolates had identical antimicrobial susceptibility patterns. Proteomic analysis revealed nine common differentially expressed proteins. Among these, the greater level of elongation factor Tu (EF-Tu) in EUK was validated by Western blotting. Outer membrane vesicles (OMVs) derived from EUK had greater promoting activities on CaOx crystallization, crystal growth and aggregation as compared to those derived from EUU. Neutralizing the OMVs of EUK with monoclonal anti-EF-Tu antibody, not with an isotype antibody, significantly reduced all these OMVs-induced promoting effects. Moreover, immunofluorescence staining of EF-Tu on bacterial cell surface confirmed the greater expression of surface EF-Tu on EUK (vs. EUU). Our data indicate that surface EF-Tu and OMVs play significant roles in promoting activities of E. coli on CaOx crystallization, crystal growth and aggregation.

Cytotoxicity of Cratoxylum Formosum Subsp. Pruniflorum Gogel Extracts in Oral Cancer Cell Lines.

BACKGROUND Oral cancer is a health problem in Thailand. Cratoxylum formosum subsp. pruniflorum Gogel (Teawdang), normally consumed in northeast Thailand, has proven cytotoxic to cervical cancer cell lines including HeLa, SiHa and C-33A. Recently, Asian oral cancer cell lines, ORL-48 and ORL-136, were established. Therefore, we aimed to study cytotoxicity of Teawdang in these. Total phenolic (TPC) and flavonoid content (TFC), and antioxidant activity of Teawdang were also determined. MATERIALS AND METHODS Teawdang was purchased from Khon Kaen market during June-October 2013. Hexane (CHE), ethyl acetate (CEE) and methanol (CME) extracts of its edible part were analyzed for TPC by the folin-ciocalteau method and for TFC by an aluminium colorimetric method. Antioxidant activity and cytotoxicity in normal Vero cells and oral cancer cells were investigated. Cell viability was assessed using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assays. RESULTS CME and CEE had higher TPC and TFC and antioxidant activity than CHE. Both CME and CEE, at 200 μg dry wt/mL, were cytotoxic to the studied oral cancer cell lines. However, CME was cytotoxic to Vero cells whereas CEE was not. Compared to Vero cells, CEE significantly inhibited ORL-48 and ORL-136 growth (p=0.03 and p=0.02, respectively). CONCLUSIONS CEE exhibited cytotoxic effects on the studied oral cancer cell lines but not normal Vero cells. The bioactive compounds in CEE should be further purified and elucidated for their mechanisms of action for development as anticancer agents.