Using Synthetic Biology methods to construct a functional estrogen biosensor based on split Nanoluciferase activity

The presence of estrogenic compounds (endocrine-disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time-consuming techniques such as liquid chromatography-mass spectrometry and high-performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our approach to the design of an estrogen responsive element uses methodology developed in the Wells lab (Elledge et al, 2021) to detect SARS-CoV-2 antibodies. This methodology takes advantage of the split Nanoluciferase (spLUC) protein divided into two functional domains (designated SmBit and LgBit). Based on rational engineering design we express dimerization dependent LgBit and SmBit fused to the Estrogen Receptor alpha protein (ERalpha) in bacteria cells. These two monomeric proteins will dimerize in the presence of estrogen, reconstitute the split luciferase enzyme and reestablish enzyme activity. Cells can be lysed, and luminescence detected to quantify estrogen present in the sample. We present here the construction strategy and proof of concept data demonstrating the efficiency of this dual-functional biosensor and its effectiveness for detection of estrogenic compounds in contaminated water. NSF-REU-1852150, REU Site: A multisite REU in Synthetic Biology, 2019.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Triclosan and Its Consequences on the Reproductive, Cardiovascular and Thyroid Levels.

Hygiene is essential to avoid diseases, and this is thanks to daily cleaning and disinfection habits. Currently, there are numerous commercial products containing antimicrobial agents, and although they are efficient in disinfecting, it is still not known the effect of the constant use of these products on human health. In fact, a massive use of disinfectants has been observed due to COVID-19, but the possible adverse effects are not yet known. Triclosan is one of the antimicrobial agents used in cosmetic products, toothpaste, and disinfectants. This compound is an endocrine disruptor, which means it can interfere with hormonal function, with its estrogenic and androgenic activity having already been stated. Even if the use of triclosan is well-regulated, with the maximum allowed concentration in the European Union of 0.3% (m/m), its effects on human health are still uncertain. Studies in animals and humans suggest the possibility of harmful health outcomes, particularly for the reproductive system, and in a less extent for the cardiovascular and thyroid functions. Thus, the purpose of this review was to analyse the possible implications of the massive use of triclosan, mainly on the reproductive and cardiovascular systems and on the thyroid function, both in animals and humans.

The prediabetes and gestational diabetes management: the role of lifestyle medicine in pre and post pregnancy outcomes

Background: In the women, between 24-45 y.o., during last decade, are increased the cases of undiagnosed prediabetes conditions and the gestational diabetes, lifestyle related. The diabetes education and management of related chronic conditions, in preconception and in early pregnancy, improve the quality of women's fertility and the health before and during pregnancy, and in early life. Aim: The program considered an early counselling, about antenal care, family risks of diabetes, nutritional support, obesity and weight management, hormonal balance, mental health conditions and involved the patients in a personalised solution for prevent the diabetes during pregnancy or manage the GDM and neonatal outcomes. Method: In 2018-2021 “FUTURA PROJECT” involved 440 women, divided after survey, into three groups: group 1 of 180 women with suspected prediabetes and hypertension, unhealthy lifestyle (smoke, inactivity, irregular sleep), fertility problems, group 2 of 140 women without chronic and pre diabetes conditions, but with stress, and unbalanced diet and group 3 of 120 women in pregnancy, with blood pressure problems and prenatal diabetes and GDM conditions. The program helped women in their reproductive health, with personalised program that reveals the reversible conditions of many chronic disorders due to bad lifestyle, and often undiagnosed. Particularly the diagnosis of prediabetes during preconceptional time and during early weeks of pregnancy, are often associated with hypertension and vascular risks, insulin resistance, undiagnosed genetic predisposition, weight disorders. The first part of program based on the division of three groups, after survey. The second part based on the personalised nutrition: the Mediterranean diet, with fibers, antioxidants, good fats, introducing the healthy foods with anti-inflammatory properties, and personalised physical activities planning and healthy habits management. The daily telemedicine check controls the evolution of the program. Results: The 3 groups are improved the personal lifestyle, with important results. In the group 1, after 6 months, the improvement of weight and physical activities increased the possibility of conceptions without complications, with reduction of insulin resistance, PCOS problem and oocytes' quality, and risks of endocrine disruptors. In the group 2 the health education and balanced heathy diet improved the hormonal problems, and undernutrition with hypovitaminosis (vit B group, vit. D, minerals), fundamental for the conception and early phases of fetal development. In the group 3 the management of chronic conditions improved a quality of prenatal health and the consequences of diabetes during and after pregnancy, for the mother and for the fetus, reduced risks of preeclampsia, birth defects, IUGR, hypertension, hyperglycemia post-partum, overweight during early life of children and risks of cardiovascular disease and diabetes type 2 during next 5 years in the mothers. The monitoring with telemedicine support helped or follow up and counselling during COVID-19 emergency. Discussion: The early diagnosis of prediabetes type 2 and gestational diabetes in women, with almost three risks factors as antenatal care history, overweight or obesity, fertility problems, PCOS, hypertension, unhealthy lifestyle, improve the quality of management of diabetes and its consequences in women's health, aged 24-45, and improve the women's health outcomes after birth and early life metabolic consequences.

SIXTEEN YEAR LONGITUDINAL STUDY OF U.S. SPERM DONORS CONTINUES TO DEMONSTRATE DECLINING SPERM COUNT AND MOTILITY

OBJECTIVE: It has been suggested that geographic variations in environmental toxins may impact sperm quality. Previously, we published findings that demonstrated a decline in sperm quality over an eleven year period across six regions of the United States (US).1 The current study investigates whether there was a decrease in sperm quality from a diverse set of US sperm donors across six regions in the US over a sixteen year period. Additionally, this study sought to evaluate changes between semen analysis (SA) parameters in an era of sperm collection during the COVID-19 pandemic. MATERIALS AND METHODS: Semen analyses (SA) from sperm donors aged 19-38, with 2-5 days abstinence, from 9 different geographic regions from January 2005-April 2021 were examined. The sperm donors originated from one of the following regions: Palo Alto, Los Angeles, Westwood, International Nordic Cryo Bank Denmark, Indianapolis, Cambridge, New York, Houston, and Spokane, WA. Donation date, BMI, and geographic region were recorded. Data was analyzed as a whole as well as by individual region. Primary outcomes were ejaculate volume (mL), average concentration (M/mL), motility (%), and total motile count (M). Data was analyzed using a general estimate equation (GEE) model with an exchangeable working correlation structure to account for repeated measures. RESULTS: A total of 176,706 SA specimens (from 3,532 unique donors) were analyzed. Controlling for BMI, there was a significant decline in average concentration (M/mL) (β=-1.89, p<0.0001), sperm motility (%) (β=-0.2892, p<0.0001) and total motile sperm (M) (β=-4.53, p<0.0001) over the 16-year study period. There were significant decreases in SA parameters within all geographic regions (Spokane only had two unique donors and could not be examined). Indianapolis showed a significant decrease in sperm concentration and total motile sperm, but also displayed an increase in sperm motility over the study period. CONCLUSIONS: Time related decline in sperm quality continues to be evident at a national level in young, healthy sperm donors. There was a decline across all geographic regions in all parameters except for ejaculate volume. How this decline in sperm counts impacts fertility has yet to be determined. Our modern environment involves increased exposures to endocrine disruptors and changes to lifestyle (including smoking, diet, and stress) that are postulated to impair male fertility by interfering with spermatogenesis. While a causative link to these risk factors remains to be elucidated further studies are necessary to evaluate whether this temporal decline in sperm count correlates with decreased fecundity. IMPACT STATEMENT: Sperm concentration, motility, and total motile sperm continue to decline across young, healthy sperm donors across the continental United States.