Impedance spectroscopy measurements as a tool for distinguishing different luminal content during bolus transit studies.

BACKGROUND: Intraluminal electrical impedance is a well-known diagnostic tool used to study bolus movement in the human esophagus. However, it is use in the human colon it is hindered by the fact that the content cannot be controlled and may include liquid, gas, solid, or a mixture of these at any one time. This article investigates the use of complex impedance spectroscopy to study different luminal content (liquid and gas). METHODS: An excised section of guinea pig proximal colon was placed in an organ bath with Krebs solution at 37°C and a custom built bioimpedance catheter was placed in the lumen. Liquid (Krebs) and gas (air) content was pumped through the lumen and the intraluminal impedance was measured at five different frequencies (1, 5.6, 31.6, 177.18 kHz and 1 MHz) at 10 samples per second. A numerical model was created to model the passage of bolus with different content and compared to the experimental data. KEY RESULTS: Differences in mean impedance magnitude and phase angle were found (from 1 to 177.18 kHz) for different contents. The numerical results qualitatively agreed with those in the experimental study. Conductivities of bolus had an effect on detecting its passage. CONCLUSIONS & INFERENCES: Complex impedance spectroscopy can distinguish between different luminal content within a range of measuring frequencies. The numerical model showed the importance of bolus conductivities for bolus transit studies in those where the bolus is controlled.

Discriminating movements of liquid and gas in the rabbit colon with impedance manometry.

BACKGROUND: High-resolution impedance manometry is a technique that is well established in esophageal motility studies for relating motor patterns to bolus flow. The use of this technique in the colon has not been established. METHODS: In isolated segments of rabbit proximal colon, we recorded motor patterns and the movement of liquid or gas boluses with a high-resolution impedance manometry catheter. These detected movements were compared to video recorded changes in gut diameter. Using the characteristic shapes of the admittance (inverse of impedance) and pressure signals associated with gas or liquid flow we developed a computational algorithm for the automated detection of these events. KEY RESULTS: Propagating contractions detected by video were also recorded by manometry and impedance. Neither pressure nor admittance signals alone could distinguish between liquid and gas transit, however the precise relationship between admittance and pressure signals during bolus flow could. Training our computational algorithm upon these characteristic shapes yielded a detection accuracy of 87.7% when compared to gas or liquid bolus events detected by manual analysis. CONCLUSIONS & INFERENCES: Characterizing the relationship between both admittance and pressure recorded with high-resolution impedance manometry can not only help in detecting luminal transit in real time, but also distinguishes between liquid and gaseous content. This technique holds promise for determining the propulsive nature of human colonic motor patterns.

High-resolution colonic motility recordings in vivo compared with ex vivo recordings after colectomy, in patients with slow transit constipation.

BACKGROUND: The pathogenesis of slow transit constipation (STC) remains poorly understood, with intrinsic and extrinsic abnormalities implicated. Here, we present high-resolution colonic manometry recordings from four STC patients recorded before total colectomy, and subsequently, ex vivo, after excision. METHODS: In four female, treatment-resistant STC patients (median age 35.5 years), a fiber-optic manometry catheter (72 sensors spaced at 1 cm intervals) was placed with the aid of a colonoscope, to the mid-transverse colon. Colonic manometry was recorded 2 h before and after a meal. After the colectomy, ex vivo colonic manometry was recorded in an organ bath. Ex vivo recordings were also made from colons from 4 patients (2 male; median age 67.5 years) undergoing anterior resection for nonobstructive carcinoma ('control' tissue). KEY RESULTS: A large increase in 'short single propagating contractions' was recorded in STC colon ex vivo compared to in vivo (ex vivo 61.3 ± 32.7 vs in vivo 2.5 ± 5/h). In STC patients, in vivo, the dominant frequency of contractile activity was 2-3 cycle per minute (cpm), whereas 1-cpm short-single propagating contractions dominated ex vivo. This same 1-cpm frequency was also dominant in control colons ex vivo. CONCLUSIONS & INFERENCES: In comparison to control adults, the colon of STC patients demonstrates significantly less propagating motor activity. However, once the STC colon is excised from the body it demonstrates a regular and similar frequency of propagating activity to control tissue. This paper provides interesting insights into the control of colonic motor patterns.

Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.

Microencapsulation of probiotic bacteria Lactobacillus plantarum 15HN using alginate-psyllium-fenugreek polymeric blends.

AIMS: Investigation on the use of herbal-based biopolymers for probiotic-Lactobacillus plantarum 15HN-encapsulation is presented. The objectives are to enhance its oral delivery, colonic release and survival rate of these probiotic cultures in gastrointestinal environment. METHODS AND RESULTS: Nine types of herbal-based polymers blend with different concentration of alginate alone or mixed with psyllium and fenugreek was used as candidate for encapsulation matrix by applying a simple extrusion method. All the blend formulations recorded high encapsulation efficiency at value >98%. The survival rate of viable probiotic cells under both low pH and high bile salt conditions was also high with value above 80% in 2% (w/v) alginate, alginate+psyllium (1·5 + 0·5%) blend and alginate+fenugreek (1·5 + 0·5%) blend as compared to other polymer formulations and nonencapsulated cells. Their release occurred after 2 h in colonic condition and sustained until the 12th hour incubation period. A value added prebiotic effect was observed in (1·5 + 0·5%) alginate-psyllium formulation. CONCLUSIONS: The high encapsulation efficiency, high viability of cell in low pH, high bile salt and the sustained release rates of probiotic cells in colonic condition during storage time was also observed for these herbal gel formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Herbal-based biopolymers offer added advantages of being prebiotic towards the enhancement of probiotic bacterial growth in the gastrointestinal environment.

Preliminary observation on the effect of baking soda volume on controlling odour from discarded organic waste.

Food wastes with high moisture and organic matter content are likely to emit odours as a result of the decomposition process. The management of odour from decomposing wastes is needed to sustain the interest of residents and local councils in the source separation of kitchen wastes. This study investigated the potential of baking soda (at 50 g, 75 g and 100g per kg food waste) to control odour from seven days stored food waste. It was found that 50 g of baking soda, spread at the bottom of 8l food wastes bin, can reduce the odour by about 70%. A higher amount (above 100g) is not advised as a pH higher than 9.0 may be induced leading to the volatilization of odorous ammonia. This research finding is expected to benefit the waste management sector, food processing industries as well as the local authorities where malodour from waste storage is a pressing issue.

Immunoproteomic analysis of antibody response to cell wall-associated proteins of Candida tropicalis.

AIMS: This study was conducted to identify antigenic proteins of Candida tropicalis that are targeted by the host immune system. METHODS AND RESULTS: An immunoproteomic approach was used to discover antigens from cell wall of C. tropicalis that were recognized by sera from experimentally infected mice. This resulted in the identification of twelve distinct proteins, of which ten have been previously reported as antigens of Candida albicans. For the remaining two proteins, Idh2p has been described as an antigen of Candida parapsilosis, whereas Kgd2p is revealed for the first time as an antigenic protein for Candida species. These two antigens were expressed as recombinant proteins in Escherichia coli and were shown to be specifically recognized by sera from infected host on Western blot. CONCLUSIONS: The present work investigated immunoproteome of C. tropicalis and identified several biomarker candidate antigens, with Kgd2p as a novel immunogenic protein that could be associated with pathogenesis of C. tropicalis. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study help to improve current understanding on host response to C. tropicalis infection and provide new insights into immune-pathogenesis of C. tropicalis. Besides, the immunogenic proteins could be considered as targets for the development of immunodiagnostic assay and/or vaccine.

A newly isolated probiotic Enterococcus faecalis strain from vagina microbiota enhances apoptosis of human cancer cells.

AIMS: This study aimed to describe probiotic properties and bio-therapeutic effects of newly isolated Enterococcus faecalis from the human vaginal tract. METHODS AND RESULTS: The Enterococcus faecalis strain was originally isolated from the vaginal microbiota of Iranian women and was molecularly identified using 16SrDNA gene sequencing. Some biochemical methodologies were preliminarily used to characterize the probiotic potential of Ent. faecalis, including antibiotic susceptibility, antimicrobial activity, as well as acid and bile resistance. The bio-therapeutic effects of this strain's secreted metabolites on four human cancer cell lines (AGS, HeLa, MCF-7 and HT-29) and one normal cell line (HUVEC) were evaluated by cytotoxicity assay and apoptosis scrutiny. The characterization results demonstrated into the isolated bacteria strain revealed probiotic properties, such as antibiotic susceptibility, antimicrobial activity and resistance under conditions similar to those in the gastrointestinal tract. Results of bio-therapeutic efficacy assessments illustrated acceptable apoptotic effects on four human cancer cell lines and negligible side effects on assayed normal cell line. Our findings revealed that the apoptotic effect of secreted metabolites mainly depended on proteins secreted by Ent. faecalis on different cancer cells. These proteins can induce the apoptosis of cancer cells. CONCLUSION: The metabolites produced by this vaginal Ent. faecalis strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells. Accordingly, the physicochemical, structural and functional properties of the secreted anticancer substances should be further investigated before using them as anticancer therapeutics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study aim to screen total bacterial secreted metabolites as a wealthy source to find the new active compounds to introduce as anticancer therapeutics in the future.

Identification of immunogenic proteins of Candida parapsilosis by serological proteome analysis.

AIMS: Systemic candidiasis is the leading fungal bloodstream infection, and its incidence has been on the rise. Recently, Candida parapsilosis has emerged as an increasingly prevalent fungal pathogen, but little is known about its antigenic profile. Hence, the current work was performed to discover immunogenic proteins of C. parapsilosis using serological proteome analysis. METHODS AND RESULTS: Cell wall proteins extracted from C. parapsilosis were resolved by two-dimensional electrophoresis followed by immunoblotting using antisera from experimentally infected mice. Mass spectrometry analysis of the 32 immunoreactive protein spots resulted in the identification of 12 distinct proteins. Among them, 11 proteins were known antigens of Candida albicans, whereas Idh2p was identified for the first time as an immunogenic protein of Candida species. Recombinant Idh2p was expressed in Escherichia coli, and its antigenicity was verified by immunoblot analysis. CONCLUSIONS: An immunoproteomic approach was successfully applied to identify immunogenic proteins of C. parapsilosis, with Idh2p as a novel candidate antigen. The identified antigens may serve as potential biomarkers for development of diagnostic assay and/or vaccine for C. parapsilosis. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents the first immunoproteomic analysis of C. parapsilosis, which provides new insights into host-pathogen interactions and pathogenesis of C. parapsilosis. The immunogenic proteins could be studied as biomarker candidates for C. parapsilosis.

Dynamics of PEGylated-dextran-spermine nanoparticles for gene delivery to leukemic cells.

Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.

Candida and invasive candidiasis: back to basics.

The ubiquitous Candida spp. is an opportunistic fungal pathogen which, despite treatment with antifungal drugs, can cause fatal bloodstream infections (BSIs) in immunocompromised and immunodeficient persons. Thus far, several major C. albicans virulence factors have been relatively well studied, including morphology switching and secreted degradative enzymes. However, the exact mechanism of Candida pathogenesis and the host response to invasion are still not well elucidated. The relatively recent discovery of the quorum-sensing molecule farnesol and the existence of quorum sensing as a basic regulatory phenomenon of the C. albicans population behavior has revolutionized Candida research. Through population density regulation, the quorum-sensing mechanism also controls the cellular morphology of a C. albicans population in response to environmental factors, thereby, effectively placing morphology switching downstream of quorum sensing. Thus, the quorum-sensing phenomenon has been hailed as the 'missing piece' of the pathogenicity puzzle. Here, we review what is known about Candida spp. as the etiological agents of invasive candidiasis and address our current understanding of the quorum-sensing phenomenon in relation to virulence in the host.

Induced pluripotent stem cells: history, properties and potential applications.

The development of induced pluripotent stem cells (iPSCs) has been met with much enthusiasm and hailed as a breakthrough discovery by the scientific and research communities amidst the divisive and ongoing debates surrounding human embryonic stem cells (hESC) research. The discovery reveals the fact that embryonic pluripotency can be generated from adult somatic cells by the induction of appropriate transcriptional factor genes essential for maintaining the pluripotency. They provide an alternative source for pluripotent stem cells, thus representing a powerful new research tool besides their potential application in the field of regenerative medicine. In this review, the historical background of iPSCs generation will be discussed together with their properties and characteristics as well as their potential therapeutic applications.

Epidemiology and strain characterization of rotavirus diarrhea in Malaysia.

OBJECTIVES: The objectives of the study were to describe the epidemiology and strain characterization of rotavirus (RV), to determine the proportion of hospitalizations for diarrhea attributable to RV among children under 5 years of age, and to estimate the disease burden of RV diarrhea in Malaysia. METHODS: All children 0-59 months of age admitted for acute gastroenteritis to Kuala Lumpur Hospital (KLH) or Hospital Umum Sarawak (HUS) were surveyed. The periods of surveillance were from February 1, 2001 to April 30, 2003 in KLH and April 1, 2001 to March 31, 2003 for HUS. RESULTS: The highest rate of RV-associated diarrhea was among children aged 6-17 months, accounting for 55% of RV-associated diarrhea. There was no seasonality observed in either hospital. P[8]G9 strains were predominant, accounting for 73% of all strains in both hospitals, 80% from KLH and 61% from HUS. There was no mortality. CONCLUSIONS: RV was responsible for 38% of hospitalizations for diarrhea. It was most common in the 6-17 months age group. There was no seasonality observed for RV-associated diarrhea. The most prevalent strain of RV was P[8]G9. The estimated incidence of RV-associated diarrhea was 27 per 10000 population under the age of 5 years per year.

The prevalence of MTHFR 677C-->T missense mutation, total plasma homocysteine levels and associated risk factors in Malay subjects.

The missense mutation of the methylenetetrahydrofolate reductase (MTHFR) gene 677C-->T is associated with modest elevation of homocysteine levels. The bio-ecogenetics factors of total homocysteine levels (tHcy) were investigated in a cross sectional study involving 53 randomly selected healthy Malay subjects. Results indicated that the prevalence of the homozygous 677T/T was 3.8% and heterozygous 677C/T was 17.0%. The levels of tHcy was higher in subjects aged more than 50 years (n = 7, 11.53 +/- 4.45 mumol/l) and in males (10.99 +/- 3.77 mumol/l) especially smoking males (12.19 +/- 3.62 mumol/l). THcy levels were low in the 3 pregnant subjects (4.44 mumol/l, p = 0.036) who were under folate supplementation.

Detection and molecular characterization of the zot gene in Vibrio cholerae and V. alginolyticus isolates.

A total of 11 Vibrio cholerae isolates from 1996-1998 outbreaks in Malaysia and 4 V. alginolyticus were analyzed. Isolates were characterized by polymerase chain reaction (PCR) and Southern hybridization for the presence of the gene encoding zonula occludens toxin (zot). Screening of zot gene by PCR revealed the presence of this gene in V. cholerae and V. alginolyticus. The zot gene from one V. cholerae Ogawa isolate that was cloned in a pCR 2.1 TOPO vector was sequenced. The sequences obtained were 99% homologous to the zot gene sequence from the Gene Bank.

Spargana infection of frogs in Malaysia.

Frogs caught from two States (Selangor and Langkawi) in Malaysia were examined for spargana of Spirometra sp. Infected frogs usually show no marks of infection but some had swelling and bleeding at the infection site. The size and weight of the infected frogs did not correlate with the infection status. The infection status in relation to human health is discussed.

Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation.

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47-phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.

Requiem: a novel zinc finger gene essential for apoptosis in myeloid cells.

To identify genes mediating programmed cell death triggered by interleukin 3 (IL-3)-deprivation of myeloid cells, the IL-3-dependent murine myeloid cell line FDCP-1 was used to screen a mammalian cell expression library for cDNAs that would promote survival following withdrawal of IL-3. A unique 892-base pair cDNA was cloned that prevented the programmed cell death response following IL-3 deprivation by causing antisense suppression of an endogenous 2.4-kilobase (kb) mRNA. A 2.3-kb cDNA containing the identical 892-base pair over-lapping sequence was cloned that encoded a deduced 371-amino acid protein containing a single Kruppel-type zinc finger and a cluster of 4 cysteine/histidine-rich repeats resembling atypical zinc fingers. The 2.4-kb mRNA was found to be ubiquitously expressed in murine tissues and its abundance in FDCP-1 cells was not altered in response to IL-3 deprivation. Since expression of this 2.4-kb mRNA was a prerequisite for the apoptosis response following IL-3 deprivation, the gene encoding it was named requiem. Requiem is likely to encode a transcription factor required for the apoptosis response following survival factor withdrawal from myeloid cells.

Antihaemophilic effect of vasopressin, deamino-(D-arginine8)-vasopressin and adrenaline in sheep: proposal for an in vivo assay system.

Female sheep were used to assay antihaemophilic (factor VIII enhancing) activity of arginine vasopressin, deamino-(D-arginine8)-vasopressin (DDAVP) and adrenaline. The time course of the response was biphasic, two surges of factor VIII being observed. DDAVP was found to be the most potent of the substances investigated. Its optimal dose was 1 microgram kg-1 body wt (i.v.). It is suggested that a similar procedure can be employed to search for new peptides with anti-haemophilic action.

[Rabies in a veterinarian]. / Tollwut bei einem Tierarzt.

A 57-year-old veterinary surgeon developed rabies 174 days after injury during an oral palpation of a rabid cow. He had been vaccinated prophylactically with Duck embryo vaccine several months before exposure but never had his serum antibody level controlled. He died 45 days later. The prodromal and encephalitic stage lasted 5 days each. Intensive supportive medical treatment was given from the beginning of the paralytic stage. The clinical course was complicated by progredient respiratory failure, diabetes insipidus, severe hypotension, and increase of intracranial pressure. The diagnosis of rabies was confirmed by rising antibody titers prior to the patient's death.