Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.     

Optical Magnetism in Surface Plasmon Resonance–Based Sensors for Enhanced Performance

Surface plasmon resonance (SPR)–based structures are finding important applications in sensing biological as well as inorganic samples. In SPR techniques, an angle-resolved reflection (R) profile of the incident light from a metal-dielectric interface is measured and the resonance characteristics are extracted for the identification of the target sample. However, the performance, and hence the applicability of these structures, suffers when the weight and concentration of the target samples are small. Here, we show that SPR-based sensors can create strong magnetism at optical frequency, which can be used for the detection of target samples instead of using the conventional R profiles, as the magnetic resonance varies depending on the refractive index of the target sample. Using scattering parameters retrieval method, we computationally find out the effective permeability (μ_eff) of a SPR sensor with a structure based on Kretschmann configuration, and use it to calculate the performance of the sensor. A comparison with the conventional technique that uses R profile to detect a target sample shows a significant increase in the sensor performance when μ_eff is used instead.

     

Function of the usher N-terminus in catalysing pilus assembly

The chaperone/usher (CU) pathway is a conserved bacterial secretion system that assembles adhesive fibres termed pili or fimbriae. Pilus biogenesis by the CU pathway requires a periplasmic chaperone and an outer membrane (OM) assembly platform termed the usher. The usher catalyses formation of subunit-subunit interactions to promote polymerization of the pilus fibre and provides the channel for fibre secretion. The mechanism by which the usher catalyses pilus assembly is not known. Using the P and type 1 pilus systems of uropathogenic Escherichia coli, we show that a conserved N-terminal disulphide region of the PapC and FimD ushers, as well as residue F4 of FimD, are required for the catalytic activity of the ushers. PapC disulphide loop mutants were able to bind PapDG chaperone-subunit complexes, but did not assemble PapG into pilus fibres. FimD disulphide loop and F4 mutants were able to bind chaperone-subunit complexes and initiate assembly of pilus fibres, but were defective for extending the pilus fibres, as measured using in vivo co-purification and in vitro pilus polymerization assays. These results suggest that the catalytic activity of PapC is required to initiate pilus biogenesis, whereas the catalytic activity of FimD is required for extension of the pilus fibre.     

Comparison of enterotoxigenic Escherichia coli isolated from surface water and diarrhoeal stool samples in Bangladesh

Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children. Although the prevalence of ETEC is high in Bangladesh and infections can be spread through food and contaminated water, limited information is available about ETEC in the surface water. We carried out studies to isolate ETEC from surface water samples from ponds, rivers, and a lake from a site close to field areas known to have a high incidence of diarrhea in Dhaka, Bangladesh, and Matlab, Bangladesh. ETEC strains isolated from the water sources were compared with ETEC strains isolated from patients with diarrhea at two hospitals in these areas. ETEC were isolated from 30% (45 of 150) of the samples from the surface water sources and 19% (518 of 2700) of the clinical specimens. One hundred ETEC strains isolated from patients with similar phenotypes as the environmental strains were compared for phenotypic and genotypic properties. The most common O serogroups on ETEC were O6, O25, O78, O115, and O126 in both types of strains. Pulsed-field gel electrophoresis analyses of the ETEC strains showed that multiple clones of ETEC were present within each colonization factor type and that some clones detected in the environment were also isolated from the stools of patients. The strains showed multiple and similar antibiotic resistance patterns. This study shows that ETEC is prevalent in surface water sources in Bangladesh suggesting a possible reason for the endemicity of this pathogen in Bangladesh.     

Bovine lactoferrin protects lipopolysaccharide-induced diarrhea modulating nitric oxide and prostaglandin E2 in mice

Lactoferrin (Lf), an iron-binding multifunctional glycoprotein, is abundantly present in colostrum and milk of different species such as humans, bovines, and mice. Our previous observation revealed that bovine colostral Lf is transported into the systemic circulation and cerebrospinal fluid from gut-lumen through receptor-mediated transcytosis in calves. Diarrhea caused by Escherichia coli is one of the important causes of infant morbidity and mortality in developing countries. We investigated the effects of bovine lactoferrin (BLf) on lipopolysaccharide (LPS)-induced diarrheogenic activity, gastrointestinal transit (GIT), and intestinal fluid content in mice. LPS accumulated abundant fluid in the small intestine in a dose-dependent manner, induced diarrhea, but decreased the GIT. Pretreatment with BLf significantly attenuated the effects of LPS on the diarrheogenic activity and intestinal content, but reversed the GIT when compared with NG-nitro-L-arginine-methyl ester (L-NAME, a non-selective NOS inhibitor) or indomethacin (an inhibitor of prostaglandin synthesis). Both plasma NO and PGE2 in enterocytes were found to increase in LPS-treated mice and were reversed by BLf. These findings demonstrate that the action of BLf against LPS was specific and it exerts antidiarrheal activity through modulating the cyclooxygenase [NO and PGE2] pathway in the gut.     

Sensitivity Analysis of Gold Nanorod Biosensors for Single Molecule Detection

Single protein molecule detection is important for investigating molecular behavior and diagnosing diseases at an early stage. Gold nanorod (GNR) biosensors have shown promise for label-free detection of single protein molecules. However, for widespread applications of GNR biosensors with high sensitivity, detail studies are needed to understand the effects of the sensing environment and the molecular binding dynamics on the sensitivity. In this work, a comprehensive theoretical analysis with variable substrate, buffer, ligand, and binding position of the target molecules shows that GNR biosensors are highly sensitive for single molecule detection of biological samples including critical pathogens such as cancer marker thyroglobulin and human immunodeficiency virus (HIV) marker glycoprotein. We also propose and show that a GNR biosensor with a dielectric cladding layer on the body increases the sensitivity by orders of magnitude compared to other state-of-the-art biosensors.

     

Phenotypic and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Bangladesh

Background

Resistance to cephalosporins in Enterobacteriaceae is mainly due to the production of extended-spectrum beta-lactamase (ESBL). Little is known about ESBL-producing bacteria in Bangladesh. Therefore, the study presents results of phenotypic and molecular characterization of ESBL-producing Escherichia coli from hospitals in Bangladesh.

Methods

A total of 339 E. coli isolated from patients with urinary tract and wound infections attending three different medical hospitals in urban and rural areas of Bangladesh between 2003–2007 were screened for ESBL-production by the double disk diffusion test. Isolates with ESBL-phenotype were further characterized by antibiotic susceptibility testing, PCR and sequencing of different β-lactamase and virulence genes, serotyping, and XbaI-macrorestriction followed by pulsed-field gel electrophoresis (PFGE).

Results

We identified 40 E. coli with ESBL phenotype. These isolates were resistant to ceftriaxone, ceftazidime, cefotaxime, aztreonam, cefepime, and nalidixic acid but remained susceptible to imipenem. All but one isolate were additionally resistant to ciprofloxacin, and 3 isolates were resistant to cefoxitin. ESBL genes of blaCTX-M-1-group were detected in all isolates; blaTEM-type and blaOXA-1-type genes were detected in 33 (82.5%) and 19 (47.5%) isolates, respectively. Virulence genes that are present in diarrhoeagenic E. coli were not found. Class-1 integron was present in 20 (50%) isolates. All the ESBL-producing E. coli isolates harbored plasmids ranging between 1.1 and 120 MDa. PFGE-typing revealed 26 different pulsotypes, but identical pulsotype showed 6 isolates of serotype O25:H4.

Conclusion

The prevalence of multidrug-resistant ESBL-producing E. coli isolates appears to be high and the majority of the isolates were positive for bla _CTX-M. Although there was genetic heterogeneity among isolates, presence of a cluster of isolates belonging to serotype O25:H4 indicates dissemination of the pandemic uropathogenic E. coli clone in Bangladesh.     

Prevalence and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx₁ and/or stx₂, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx₂, eae, katP, etpD, and enterohemorrhagic E. coli hly (hly_EHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx₁. Only 7.0% (n = 5) of the isolates were positive for hly_EHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf_O113, saa, lpfA_O157/01-141, and lpfA_O157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.     

Functional characterization, localization, and molecular identification of rabbit intestinal N-amino acid transporter

We have characterized the Na-glutamine cotransporter in the rabbit intestinal crypt cell brush border membrane vesicles (BBMV). Substrate specificity experiments showed that crypt cell glutamine uptake is mediated by system N. Real-time PCR experiments showed that SN2 (SLC38A5) mRNA is more abundant in crypt cells compared with SN1 (SLC38A3), indicating that SN2 is the major glutamine transporter present in the apical membrane of the crypt cells. SN2 cDNA was obtained by screening a rabbit intestinal cDNA library with human SN1 used as probe. Rabbit SN2 cDNA encompassed a 473-amino-acid-long open reading frame. SN2 protein displayed 87% identity and 91% similarity to human SN2. Functional characterization studies of rabbit SN2 were performed by using vaccinia virus-mediated transient expression system. Substrate specificity of the cloned transporter was identical to that of SN2 described in the literature and matched well with substrate specificity experiments performed using crypt cell BBMV. Cloned rabbit SN2, analogous to its human counterpart, is Li(+) tolerant. Hill coefficient for Li(+) activation of rabbit SN2-mediated uptake was 1. Taken together, functional data from the crypt cell BBMV and the cloned SN2 cDNA indicate that the crypt cell glutamine transport is most likely mediated by SN2.