Bioengineered bioluminescent magnetotactic bacteria as a powerful tool for chip-based whole-cell biosensors

This paper describes the generation of genetically engineered bioluminescent magnetotactic bacteria (BL-MTB) and their integration into a microfluidic analytical device to create a portable toxicity detection system. Magnetospirillum gryphiswaldense strain MSR-1 was bioengineered to constitutively express a red-emitting click beetle luciferase whose bioluminescent signal is directly proportional to bacterial viability. The magnetic properties of these bacteria have been exploited as "natural actuators" to transfer the cells in the chip from the reaction to the detection area, optimizing the chip's analytical performance. A robust and cost-effective biosensor for the evaluation of sample toxicity, named MAGNETOX, based on lens-free contact imaging detection, has been developed. A microfluidic chip has been fabricated using multilayered black and transparent polydimethyl siloxane (PDMS) in which BL-MTB are incubated for 30 min with the sample, then moved by microfluidics, trapped, and concentrated in detection chambers by an array of neodymium-iron-boron magnets. The chip is placed in contact with a cooled CCD via a fiber optic taper to perform quantitative bioluminescence imaging after addition of luciferin substrate. A model toxic compound (dimethyl sulfoxide, DMSO) and a bile acid (taurochenodeoxycholic acid, TCDCA) were used to investigate the analytical performance of the MAGNETOX. Incubation with DMSO and TCDCA drastically reduces the bioluminescent signal in a dose-related manner. The generation of bacteria that are both magnetic and bioluminescent combines the advantages of easy 2D cell handling with ultra sensitive detection, offering undoubted potential to develop cell-based biosensors integrated into microfluidic chips

A novel bioluminescent NanoLuc yeast-estrogen screen biosensor (nanoYES) with a compact wireless camera for effect-based detection of endocrine-disrupting chemicals

The presence of chemicals with estrogenic activity in surface, groundwater, and drinking water poses serious concerns for potential threats to human health and aquatic life. At present, no sensitive portable devices are available for the rapid monitoring of such contamination. Here, we propose a cell-based mobile platform that exploits a newly developed bioluminescent yeast-estrogen screen (nanoYES) and a low-cost compact camera as light detector. Saccharomyces cerevisiae cells were genetically engineered with a yeast codon-optimized variant of NanoLuc luciferase (yNLucP) under the regulation of human estrogen receptor α activation. Ready-to-use 3D-printed cartridges with immobilized cells were prepared by optimizing a new procedure that enables to produce alginate slices with good reproducibility. A portable device was obtained exploiting a compact camera and wireless connectivity enabling a rapid and quantitative evaluation (1-h incubation at room temperature) of total estrogenic activity in small sample volumes (50 μL) with a LOD of 0.08 nM for 17β-estradiol. The developed portable analytical platform was applied for the evaluation of water samples spiked with different chemicals known to have estrogen-like activity. Thanks to the high sensitivity of the newly developed yeast biosensor and the possibility to wireless connect the camera with any smartphone model, the developed configuration is more versatile than previously reported smartphone-based devices, and could find application for on-site analysis of endocrine disruptors. [Figure not available: see fulltext.]

Effect of Colic Vein Ligature in Rats with Loperamide-Induced Constipation

Introduction. Medical treatment in chronic constipation is not always successful. Surgery is indicated in unresponsive selected severe cases. This study presents the distal venous colic ligation in rat as a novel surgical approach. Materials and Methods. 16 rats (study group) were evaluated in 3 phases of 6 days each: A (normal conditions), B (loperamide-induced constipation), and C (colic vein legation) and compared with rats treated in phase C with PEG 4,000 (control group). Blood biochemical and physiological parameters, daily fecal water content (FWC), and histological analysis were performed in all study phases. Results. No biochemical and physiological parameters changes were observed. FWC decreased in phase B and increased in phase C in both groups with a grow up to 2.3-fold in study group compared to control (P < 0.0001). Moreover, in study group, a high number of colonic goblet cells were detected (phase C versus phase B: P < 0.001) while no differences were registered in control. Conclusion. By ligature of the colic vein in constipated rats, an increase in FWC and goblet cells higher than in PEG treated rats was detected. The described surgical procedure appeared effective, simple, and safe; further studies in animal models, however, are necessary to assess its clinical applicability

Smartphone-based multicolor bioluminescent 3D spheroid biosensors for monitoring inflammatory activity

Whole-cell biosensors present many advantages, including being able to monitor the toxicity and bioavailability of chemicals; cells grown in traditional 2D cultures, however, do not reproduce the complexity of in vivo physiology. In the last years, 3D cell-culture models have garnered great attention due to their capability to better mimic in vivo cellular responses to external stimuli, providing excellent model living organisms. In order to obtain a predictive, sensitive, and robust yet low-cost 3D cell biosensor, we developed a smartphone-based bioluminescent 3D cell biosensor platform for effect-based analysis. We exploited the Nuclear Factor-kappa B (NF-kB) signal transduction pathway, which is induced by several types of stressors and is involved in the regulation of cell-cycle/growth, inflammation, apoptosis, and immunity. The smartphone-based biosensor relies on immobilized HEK293 spheroids genetically engineered with powerful red- and green-emitting luciferases utilized as inflammation and viability reporters. It provides a limit of detection for Tumor Necrosis Factor (TNF\u3b1) of 0.15\u202f\ub1\u202f0.05\u202fng/mL and could be a useful tool to initially screen environmental samples or other compounds on-site, especially for additional more accurate chemical analyses

Insights Into Sibling Relationships and Longevity From Genetics of Healthy Ageing Nonagenarians: The Importance of Optimisation, Resilience and Social Networks

Understanding how to “Age Longer and Age Well” is a priority for people personally, for populations and for government policy. Approximately ten percent of nonagenarians reach 90 years and beyond in good condition and seem to have a combination of both age-span and health-span. However, the factors which contribute to human longevity remain challenging. Culture is a shared system of learning ideas, feelings, and survival strategies. It has a strong influence on each person’s psychological development, behavior, values and beliefs. Nonagenarians have rich life experiences that can teach us much about aging well; they are rich reservoirs of genetic, lifestyle and psychological information which can help understanding about how to live longer and better. Sibling or trio nonagenarians are important sources of family beliefs and behaviors upon which individual personalities may have been built. Their personal family histories and narratives are powerful tools that help to determine familial traits, beliefs and social behaviors which may help establish factors important in the siblings’ longevity. Using purposefully selected subjects, recruited to the Genetics of Healthy Ageing (GeHA) project in four European countries, this research used the simple life story and qualitative research methods to analyze contrasting and distinctive questions about the interface between the psychological and social worlds as presented in the nonagenarian siblings’ insights about their longevity. Their stories aimed to give better understanding about which psychological aspects of their common life journey and the degree of emotional support in their sibling relationships may have supported their paths to longevity. The most universal finding in each of the four European countries was that nonagenarians demonstrated high positivity, resilience and coping skills and were supported in social networks. Around this theme, nonagenarians reported “being happy,” “always cheerful,” “never melancholy” and having a contentment with a “rich life” and family relationships which fits with accumulating evidence that life satisfaction comes from a perceived self-efficacy and optimism. Most sibling relationships in this study, when analyzed according to the Gold classification, fit the “congenial” or “loyal” relationship type – demonstrating a healthy respect for the others’ opinion without overt dependence, which may help individual coping and survival mechanisms

Effect of Colic Vein Ligature in Rats with Loperamide-Induced Constipation

Introduction. Medical treatment in chronic constipation is not always successful. Surgery is indicated in unresponsive selected severe cases. This study presents the distal venous colic ligation in rat as a novel surgical approach. Materials and Methods. 16 rats (study group) were evaluated in 3 phases of 6 days each: A (normal conditions), B (loperamide-induced constipation), and C (colic vein legation) and compared with rats treated in phase C with PEG 4,000 (control group). Blood biochemical and physiological parameters, daily fecal water content (FWC), and histological analysis were performed in all study phases. Results. No biochemical and physiological parameters changes were observed. FWC decreased in phase B and increased in phase C in both groups with a grow up to 2.3-fold in study group compared to control (P &lt; 0.0001). Moreover, in study group, a high number of colonic goblet cells were detected (phase C versus phase B: P &lt; 0.001) while no differences were registered in control. Conclusion. By ligature of the colic vein in constipated rats, an increase in FWC and goblet cells higher than in PEG treated rats was detected. The described surgical procedure appeared effective, simple, and safe; further studies in animal models, however, are necessary to assess its clinical applicability

The Integrated European Project "GEHA - GEnetics of Healthy Aging": recruitment, health status assessment and survival of the Italian 90+ sibpairs

The present study is part of the EU Integrated Project “GEHA – Genetics of Healthy Aging” (Franceschi C et al., Ann N Y Acad Sci. 1100: 21-45, 2007), whose aim is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced age in good cognitive and physical function and in the absence of major age-related diseases. Aims The major aims of this thesis were the following: 1. to outline the recruitment procedure of 90+ Italian siblings performed by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The procedures related to the following items necessary to perform the study were described and commented: identification of the eligible area for recruitment, demographic aspects related to the need of getting census lists of 90+siblings, mail and phone contact with 90+ subjects and their families, bioethics aspects of the whole procedure, standardization of the recruitment methodology and set-up of a detailed flow chart to be followed by the European recruitment centres (obtainment of the informed consent form, anonimization of data by using a special code, how to perform the interview, how to collect the blood, how to enter data in the GEHA Phenotypic Data Base hosted at Odense). 2. to provide an overview of the phenotypic characteristics of 90+ Italian siblings recruited by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The following items were addressed: socio-demographic characteristics, health status, cognitive assessment, physical conditions (handgrip strength test, chair-stand test, physical ability including ADL, vision and hearing ability, movement ability and doing light housework), life-style information (smoking and drinking habits) and subjective well-being (attitude towards life). Moreover, haematological parameters collected in the 90+ sibpairs as optional parameters by the Bologna and Rome recruiting units were used for a more comprehensive evaluation of the results obtained using the above mentioned phenotypic characteristics reported in the GEHA questionnaire. 3. to assess 90+ Italian siblings as far as their health/functional status is concerned on the basis of three classification methods proposed in previous studies on centenarians, which are based on: • actual functional capabilities (ADL, SMMSE, visual and hearing abilities) (Gondo et al., J Gerontol. 61A (3): 305-310, 2006); • actual functional capabilities and morbidity (ADL, ability to walk, SMMSE, presence of cancer, ictus, renal failure, anaemia, and liver diseases) (Franceschi et al., Aging Clin Exp Res, 12:77-84, 2000); • retrospectively collected data about past history of morbidity and age of disease onset (hypertension, heart disease, diabetes, stroke, cancer, osteopororis, neurological diseases, chronic obstructive pulmonary disease and ocular diseases) (Evert et al., J Gerontol A Biol Sci Med Sci. 58A (3): 232-237, 2003). Firstly these available models to define the health status of long-living subjects were applied to the sample and, since the classifications by Gondo and Franceschi are both based on the present functional status, they were compared in order to better recognize the healthy aging phenotype and to identify the best group of 90+ subjects out of the entire studied population. 4. to investigate the concordance of health and functional status among 90+ siblings in order to divide sibpairs in three categories: the best (both sibs are in good shape), the worst (both sibs are in bad shape) and an intermediate group (one sib is in good shape and the other is in bad shape). Moreover, the evaluation wanted to discover which variables are concordant among siblings; thus, concordant variables could be considered as familiar variables (determined by the environment or by genetics). 5. to perform a survival analysis by using mortality data at 1st January 2009 from the follow-up as the main outcome and selected functional and clinical parameters as explanatory variables. Methods A total of 765 90+ Italian subjects recruited by UNIBO (549 90+ siblings, belonging to 258 families) and ISS (216 90+ siblings, belonging to 106 families) recruiting units are included in the analysis. Each subject was interviewed according to a standardized questionnaire, comprising extensively utilized questions that have been validated in previous European studies on elderly subjects and covering demographic information, life style, living conditions, cognitive status (SMMSE), mood, health status and anthropometric measurements. Moreover, subjects were asked to perform some physical tests (Hand Grip Strength test and Chair Standing test) and a sample of about 24 mL of blood was collected and then processed according to a common protocol for the preparation and storage of DNA aliquots. Results From the analysis the main findings are the following: - a standardized protocol to assess cognitive status, physical performances and health status of European nonagenarian subjects was set up, in respect to ethical requirements, and it is available as a reference for other studies in this field; - GEHA families are enriched in long-living members and extreme survival, and represent an appropriate model for the identification of genes involved in healthy aging and longevity; - two simplified sets of criteria to classify 90+ sibling according to their health status were proposed, as operational tools for distinguishing healthy from non healthy subjects; - cognitive and functional parameters have a major role in categorizing 90+ siblings for the health status; - parameters such as education and good physical abilities (500 metres walking ability, going up and down the stairs ability, high scores at hand grip and chair stand tests) are associated with a good health status (defined as “cognitive unimpairment and absence of disability”); - male nonagenarians show a more homogeneous phenotype than females, and, though far fewer in number, tend to be healthier than females; - in males the good health status is not protective for survival, confirming the male-female health survival paradox; - survival after age 90 was dependent mainly on intact cognitive status and absence of functional disabilities; - haemoglobin and creatinine levels are both associated with longevity; - the most concordant items among 90+ siblings are related to the functional status, indicating that they contain a familiar component. It is still to be investigated at what level this familiar component is determined by genetics or by environment or by the interaction between genetics, environment and chance (and at what level). Conclusions In conclusion, we could state that this study, in accordance with the main objectives of the whole GEHA project, represents one of the first attempt to identify the biological and non biological determinants of successful/unsuccessful aging and longevity. Here, the analysis was performed on 90+ siblings recruited in Northern and Central Italy and it could be used as a reference for others studies in this field on Italian population. Moreover, it contributed to the definition of “successful” and “unsuccessful” aging and categorising a very large cohort of our most elderly subjects into “successful” and “unsuccessful” groups provided an unrivalled opportunity to detect some of the basic genetic/molecular mechanisms which underpin good health as opposed to chronic disability. Discoveries in the topic of the biological determinants of healthy aging represent a real possibility to identify new markers to be utilized for the identification of subgroups of old European citizens having a higher risk to develop age-related diseases and disabilities and to direct major preventive medicine strategies for the new epidemic of chronic disease in the 21st century

Arm measurements as determinants of further survival in centenarians

Being indicators of nutritional and functional status, anthropometric charactersmay have great prognostic signif- icance for survival at extremely advanced ages. For ethical and practical reasons however it is advisable to use characters such as armmeasurements easily measurable even in bedridden subjects. This study compares the in- fluence of some upper arm measurements and of Body Mass Index (BMI) on survival of the 77 subjects aged 98 years and over (98+) recruited within the MALVA project, one of the first Italian population-based studies on extremely old people. Adopting methods for multiple imputation of missing values, Gompertz regression models adjusted for gender and age were estimated for each anthropometric character or combination of char- acters, i.e. BMI;mid-upper armcircumference (MUAC)+ elbow breadth (EB)+ triceps skinfold thickness (TSF); corrected arm muscle area (CAMA). Being underweight and having a low CAMA and a low MUAC/high EB were positively associated with an increased risk of death, while no significant association was found with the condi- tion of being overweight/obese and the triceps skinfold thickness.When anthropometric variables were included in regression models along with covariates relating to nutritional and functional status, BMI and MUAC, but not CAMA, emerged as protective factors. It is suggested that MUAC can be recommended in evaluating the health status of extremely old people and that measuring EB may help to estimate the non-boney component of the arm