Genetically engineered filamentous phage for bacterial detection using magnetic resonance imaging.

Detecting bacterial cells with high specificity in deep tissues is challenging. Optical probes provide specificity, but are limited by the scattering and absorption of light in biological tissues. Conversely, magnetic resonance imaging (MRI) allows unfettered access to deep tissues, but lacks contrast agents for detecting specific bacterial strains. Here, we introduce a biomolecular platform that combines both capabilities by exploiting the modularity of M13 phage to target bacteria with tunable specificity and allow deep-tissue imaging using T1-weighted MRI. We engineered two types of phage probes: one for detecting the phage's natural host, viz., F-pilus expressing E. coli; and the other for detecting a different (F-negative) bacterial target, V. cholerae. We show that these phage sensors generate 3-9-fold stronger T1 relaxation upon recognizing target cells relative to non-target bacteria. We further establish a preliminary proof-of-concept for in vivo applications, by demonstrating that phage-labeled bacteria can be detected in mice using MRI. The framework developed in this study may have potential utility in a broad range of applications, from basic biomedical research to in situ diagnostics, which require methods to detect and track specific bacteria in the context of intact living systems.

A Protein-Based Biosensor for Detecting Calcium by Magnetic Resonance Imaging.

Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer a promising approach for noninvasive brain-wide monitoring of neural activity at any arbitrary depth. Current examples of MRI-based calcium probes involve synthetic molecules and nanoparticles, which cannot be used to examine calcium signaling in a genetically encoded form. Here, we describe a new MRI sensor for calcium, based entirely on a naturally occurring calcium-binding protein known as calprotectin. Calcium-binding causes calprotectin to sequester manganese ions, thereby limiting Mn2+ enhanced paramagnetic relaxation of nearby water molecules. We demonstrate that this mechanism allows calprotectin to alter T1 and T2 based MRI signals in response to biologically relevant calcium concentrations. The resulting response amplitude, i.e., change in relaxation time, is comparable to existing MRI-based calcium sensors as well as other reported protein-based MRI sensors. As a preliminary demonstration of its biological applicability, we used calprotectin to detect calcium in a lysed hippocampal cell preparation as well as in intact Chinese hamster ovary cells treated with a calcium ionophore. Calprotectin thus represents a promising path toward noninvasive imaging of calcium signaling by combining the molecular and cellular specificity of genetically encodable tools with the ability of MRI to image through scattering tissue of any size and depth.

Calcium-responsive contrast agents for functional magnetic resonance imaging.

Calcium ions represent one of the key second messengers accompanying neural activity and synaptic signaling. Accordingly, dynamic imaging of calcium fluctuations in living organisms represents a cornerstone technology for discovering neural mechanisms that underlie memory, determine behavior, and modulate emotional states as well as how these mechanisms are perturbed by neurological disease and brain injury. While optical technologies are well established for high resolution imaging of calcium dynamics, physical limits on light penetration hinder their application for whole-brain imaging in intact vertebrates. Unlike optics, magnetic resonance imaging (MRI) enables noninvasive large-scale imaging across vertebrates of all sizes. This has motivated the development of several sensors that leverage innovative physicochemical mechanisms to sensitize MRI contrast to intracellular and extracellular changes in calcium. Here, we review the current state-of-the-art in MRI-based calcium sensors, focusing on fundamental aspects of sensor performance, in vivo applications, and challenges related to sensitivity. We also highlight how innovations at the intersection of reporter gene technology and gene delivery open potential opportunities for mapping calcium activity in genetically targeted cells, complementing the benefits of small molecule probes and nanoparticle sensors.

Beyond the Green Fluorescent Protein: Biomolecular Reporters for Anaerobic and Deep-Tissue Imaging.

Fluorescence imaging represents cornerstone technology for studying biological function at the cellular and molecular levels. The technology's centerpiece is a prolific collection of genetic reporters based on the green fluorescent protein (GFP) and related analogs. More than two decades of protein engineering have endowed the GFP repertoire with an incredible assortment of fluorescent proteins, allowing scientists immense latitude in choosing reporters tailored to various cellular and environmental contexts. Nevertheless, GFP and derivative reporters have specific limitations that hinder their unrestricted use for molecular imaging. These challenges have inspired the development of new reporter proteins and imaging mechanisms. Here, we review how these developments are expanding the frontiers of reporter gene techniques to enable nondestructive studies of cell function in anaerobic environments and deep inside intact animals-two important biological contexts that are fundamentally incompatible with the use of GFP-based reporters.

Creation of a formate: malate oxidoreductase by fusion of dehydrogenase enzymes with PEGylated cofactor swing arms.

Enzymatic biocatalysis can be limited by the necessity of soluble cofactors. Here, we introduced PEGylated nicotinamide adenine dinucleotide (NAD(H)) swing arms to two covalently fused dehydrogenase enzymes to eliminate their nicotinamide cofactor requirements. A formate dehydrogenase and cytosolic malate dehydrogenase were connected via SpyCatcher-SpyTag fusions. Bifunctionalized polyethylene glycol chains tethered NAD(H) to the fusion protein. This produced a formate:malate oxidoreductase that exhibited cofactor-independent ping-pong kinetics with predictable Michaelis constants. Kinetic modeling was used to explore the effective cofactor concentrations available for electron transfer in the complexes. This approach could be used to create additional cofactor-independent transhydrogenase biocatalysts by swapping fused dehydrogenases.

Mimicking the biological olfactory system: a Portable electronic Mucosa.

In this study the authors report on the development of a new type of electronic nose (e-nose) instrument, which the authors refer to as the Portable electronic Mucosa (PeM) as a continuation of previous research. It is designed to mimic the human nose by taking significant biological features and replicating them electronically. The term electronic mucosa or simply e-mucosa was used because our e-nose emulates the nasal chromatographic effect discovered in the olfactory epithelium, located within the upper turbinate. The e-mucosa generates spatio-temporal information that the authors believe could lead to improved odour discrimination. The PeM comprises three large sensor arrays each containing a total of 576 sensors, with 24 different coatings, to increase the odour selectivity. The nasal chromatographic effect provides temporal information in the human olfactory system, and is mimicked here using two-coated retentive channels. These channels are coated with polar and non-polar compounds to enhance the selectivity of the instrument. Thus, for an unknown sample, the authors have both the spatial information (as with a traditional e-nose) and the temporal information. The authors believe that this PeM may offer a way forward in developing a new range of low-cost e-noses with superior odour specificity.

Outcome of moderate to severe malnutrition following persistent diarrhoea--a hospital-based retrospective study.

We aimed to determine the outcome of nutritional intervention in children with moderate to severe malnutrition following persistent diarrhoea (PD), referred to a tertiary referral unit in Malaysia. Thirty-one (44%) of the 71 children (median age 19 months) with PD had moderate to severe malnutrition on admission. Fifty-three (75%) required dietary modification and 15 (21%) needed parenteral nutrition (PN, median duration 96 days). Of the 70 patients in whom remission of diarrhoea could be ascertained, 64 (91%) achieved remission. Three required home PN. At three months after discharge, there was a significant improvement in the mean weight-for-height z-score as compared to the original score at initial presentation (from -1.83 +/- -1.77 to -0.80 +/- -1.17; p < 0.001), although 12 (22%) of the 55 patients in whom nutritional status could be ascertained still had moderate to severe malnutrition. In conclusion, moderate to severe malnutrition was a common complication following PD resulting from diverse causes. With appropriate therapy, remission can be achieved in majority of patients, although a small number of patients needed home PN because of persistence of diarrhoea.

A national database on children and adolescent with diabetes (e-DiCARE): results from April 2006 to June 2007.

In Malaysia, Diabetes in Children and Adolescents Registry (DiCARE) was launched nationwide in August 2006 to determine and monitor the number, the time trend of diabetes mellitus (DM) patients, their socio-demographic profiles, outcome of intervention and facilitate research using this registry. This is an on going real time register of diabetic patients < or = 20 years old via the e-DiCARE, an online registration system. To date were 240 patients notified from various states in Malaysia. The mean age was 12.51 years (1.08-19.75) and 46.4% were boys. The mean age at diagnosis was 8.31 +/- 4.13 years old with an estimated duration of diabetes of 4.32 +/- 3.55 years. A total of 166/240 (69.2%) have T1DM, 42/240 (17.5%) have T2DM and 18/240 (7.5%) have other types of DM. Basis of diagnosis was known in 162 patients with T1DM and 41 patients with T2DM. In T1DM patients, 6.0% of the girls and 19.1% boys were overweight or obese. As for T2DM, 64.3% had their BMI reported: 66.7% girls and 91.6% boys were overweight or obese. Most patients (80.4%) practiced home blood glucose monitoring. Patients were seen by dietitian (66.7%), diabetes educator (50.0%), and optometrist or ophthalmologist (45.0%). Only 10.8% attended diabetic camps. In the annual census of 117 patients, the mean HbAlc level was 10.0% + 2.2 (range 5.2 to 17.0%). The early results of DiCARE served as a starting point to improve the standard of care of DM among the young in the country.

Factors affecting nutritional status of Malaysian primary school children.

This paper investigates the nutritional status of a randomly selected cohort of school children and the factors affecting it. This random survey was conducted in the state of Selangor, involving 1,405 primary students (aged 9-10 years from 54 national primary schools). Physical examination was carried out on all the students. Information on the students was also obtained from the parents. Blood samples were taken by using the finger pricking technique. Body mass index (BMI) was used as a measure of physical growth. The students were mainly from urban areas (82.9%). The mean age was 9.71 years and a higher proportion was females (51%). Malays constituted 83.6%, Indians 11.6% and Chinese 4.2% of the study population. The mean weight and height were 32.30 kg and 135.18 cm respectively. The mean BMI was 17.42 kg/m2, with 1.2% of the students underweight, 76.3% normal BMI, 16.3% overweight and 6.3% were obese. Nutritional status was significantly related to blood pressure, history of breast feeding, eating fast food, taking canned/bottled drinks, income and educational level of parents. Significant differences in nutritional status between sexes and locations (rural/urban) were also found. The prevalence of overweight and obese children was of concern. There is thus an urgent need for the School Health Program to periodically monitor the school children's eating habits and physical growth. Appropriate counselling on nutritional intake and physical activities should be given not only to schoolchildren but also to their teachers and parents or caregivers.

Effects of nutritional status on academic performance of Malaysian primary school children.

Numerous factors are known to affect the academic performance of students. These include prenatal conditions, birth conditions, postnatal events, nutritional, socio-economic factors and environmental factors. This paper examines the nutritional status and its relationship with academic performance of 9-10 years old primary school children recruited randomly in Selangor, Malaysia. A standard self-administered questionnaire was utilized to obtain pertinent information and a face-to-face interview was also conducted with the parents. Results of the academic performances were extracted from the students' report cards. The intellectual performance was assessed using Raven's Coloured Progressive Matrices. Physical examination was also conducted on these students by doctors. Overall 1,405 students and 1,317 parents responded to the survey. Of these 83.6% were Malays, 11.6% Indians, and 4.2% Chinese. The majority of them (82.9%) were from urban areas. The female: male ratio was 51:49; mean age was 9.71 years. The mean height and weight were 32.3 kg and 135.2 cm respectively. Their mean BMI was 17.42 kg/cm2, with 0.9% underweight, 76.3% normal BMI, 16.3% overweight, and 6.3% obese. Academic performance was significantly correlated with breast feeding, income and educational level of their parents, BMI, and whether they have been taking breakfast. There was a weak correlation between presence of anaemia and intellectual performance. Improving the socio-economic status of the parents will lend a helping hand in the academic performance of the students. Since breast feeding is associated with better academic and intellectual performance it must be emphasized, particularly to expectant mothers in the antenatal clinics.

Addisonian-like crisis in congenital hypopituitarism and cholestatic jaundice.

A six-week-old male infant was admitted for investigation of cholestasis and pale stools. He became lethargic and apnoeic with prolonged seizures after a percutaneous liver biopsy. Subsequent investigations showed conjugated hyperbilirubinaemia, elevated liver enzymes, and hypoglycaemia. The radinuclide hepatobiliary scintigraphy was non-excretory. After an operative cholangiogram, the infant developed Addisonian-like crisis with bradycardia, hypotension, respiratory distress, metabolic acidosis, hypoglycaemia, hyponatraemia, and hyperkalaemia. Blood investigations confirmed congenital hypopituitarism. Hormone replacement therapy with L-thyroxine and cortisone acetate resulted in dissolution of jaundice and the reduction of the liver size.

Dengue encephalitis: a true entity?

Involvement of the central nervous system in dengue fever and dengue hemorrhagic fever has always been thought to be secondary to vasculitis with resultant fluid extravasation, cerebral edema, hypoperfusion, hyponatremia, liver failure, and/or renal failure. Thus, the condition has been referred to as dengue encephalopathy. Encephalitis or direct involvement of the brain by the virus was thought to be unlikely. This paper reports on six children who were seen over a period of two years presenting on the second or third day of illness with dengue encephalitis. The diagnosis was based upon a clinical picture of encephalitis and confirmed by cerebrospinal fluid (CSF) microscopy and electroencephalography changes. All six cases were confirmed dengue infections. Dengue 3 virus was isolated from the CSF of four cases and in one case, dengue 2 was detected by the polymerase chain reaction in both the CSF and blood. In the sixth case, virologic evidence was negative but dengue immunoglobulin M was detected in the CSF and blood. Since the onset of encephalitis appears early in the course of illness coinciding with the viremic phase, we postulate that the virus crosses the blood-brain barrier and directly invades the brain causing encephalitis. This study provides strong evidence that dengue 2 and 3 viruses have neurovirulent properties and behave similarly to other members of the Flaviviridae.

Strategy for child immunization in Malaysian plantations.

An outline is given of a simple cost-effective strategy aimed at the immunization of all children and pregnant women residing in the plantation sector of Malaysia. It is based on a partnership between government, nongovernmental organizations and the private sector, and is supported by UNICEF.

PIP: A cost-effective strategy aimed at the immunization of all children and pregnant women residing in the plantation sector of Malaysia is outlined. It is based on a partnership between government, nongovernmental organizations and the private sector, and is supported by UNICEF. Over a million people reside on the Malaysian plantation estates: only 17% of the estates have their own hospitals; immunization services exist on only 1.5%; 40% of the estates are at least 5 kilometers from the nearest government health facility; and 64% lack transport for workers and their dependents to seek care away from the plantations. Two nongovernmental organizations, the Malaysian Paediatric Association and the Malaysian Society of Health, initiated discussions with the United Planting Association of Malaysia. A pilot study was undertaken by the groups on 6 estates in Selangor State, which included all the children at their first birthday. Tuberculosis, diphtheria/pertussis/tetanus, poliomyelitis, and measles immunization coverages were 88%, 44%, 59%, and 66%, respectively. The association of plantations accepted the organizations' proposals for all estates to: register all births; provide free transportation to government health clinics for the immunization of all eligible children and pregnant women; and enforce immunization schedules and record-keeping. The Ministry of Health agreed to provide free immunization of children and pregnant women; send mobile teams to estates that could assemble 20 or more eligible people for immunization; provide the estates with educational materials dealing with immunization; arrange that the maintenance of the cold chain be supervised by local medical officers of health; consider the training of estate hospital assistants with the help of the nongovernmental organizations. The total immunization plan was launched in September 1990. A manual was distributed to the estate managers, hospital assistants on the estates, and the medical officers who would implement and monitor the program. It is expected that total child immunization will be achieved in the foreseeable future in the estate sector.