Acetylcholinesterase Biosensor Based on Functionalized Renewable Carbon Platform for Detection of Carbaryl in Food.

Enzymatic electrochemical biosensors play an important role in the agri-food sector due to the need to develop sustainable, low-cost, and easy-to-use analytical devices. Such biosensors can be used to monitor pathogens, endocrine disruptors, and pesticides, such as carbaryl, widely used in many crops. The use of renewable carbon (RC) sources, provided from biomass pyrolysis has been often applied in the fabrication of such sensors. This material is a great candidate for biosensor fabrication due to the presence of surface functional groups, porosity, and moderate surface area. This work describes the functionalization of RC material through an acid treatment with a sulfonitric solution HNO3/H2SO4 (1:3) and the resulting material was characterized by scanning electron microscopy. The obtained RC functionalized (RCF) and the acetylcholinesterase enzyme (AChE) were applied in the construction of the electrochemical biosensor on glassy carbon (GC) electrode and used to detect carbaryl in apple samples. The GC/RCF/AChE biosensor was able to detect the carbaryl pesticide from 5.0 to 30.0 nmol L-1, displaying a LOD of 4.5 nmol L-1. The detection of carbaryl in apple samples presented recoveries between 102.5 to 118.6% through the standard addition method. The proposed biosensor is a promising renewable tool for food safety.

Fabrication of paper-based analytical devices using a PLA 3D-printed stencil for electrochemical determination of chloroquine and escitalopram.

In recent years, the use of prescribed and non-prescribed drugs has increased. Therefore, advances in new technologies and sensors for detecting molecules in natural environments are required. In this work, a 3D-printed polylactic acid stencil is used to fabricate paper-based analytical devices (ePADs). Herein, we report the use of carbon-based lab-manufactured conductive ink for the fabrication of sensors towards the detection of chloroquine and escitalopram. For each batch, eight ePADs were successfully fabricated. Firstly, the fabricated sensors were evaluated morphologically by scanning electron microscopy and electrochemically by cyclic voltammetry and electrochemical impedance spectroscopy experiments. The sensors displayed a well-defined voltammetric profile in the presence of the redox couple, when compared to a commercial carbon screen-printed electrode. Differential pulse voltammetry conducted the detection of chloroquine and escitalopram with detection limits of 4.0 and 0.5 µmol L-1, respectively. The ePADs fabricated using the 3D stencil are here presented as alternatives for the fabrication of electrochemical analytical devices. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10008-021-05075-w.

A Novel Method for the Detection of SARS-CoV-2 Based on Graphene-Impedimetric Immunosensor.

Due to the SARS-CoV-2 pandemic, there has been an increase in the search for affordable healthcare devices for mass testing and rapid diagnosis. In this context, this work described a new methodology for SARS-CoV-2 detection based on an impedimetric immunosensor developed using the advantageous immobilization of antibodies in the reduced graphene oxide (rGO). The rGO was obtained by chemical synthesis from the commercial graphene oxide (GO), and the materials were morphologically, electrochemically and visually characterized. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were used to evaluate the fabrication steps of the immunosensor. The electrochemical immunoassay was considered for SARS-CoV-2 spike protein RBD detection using a impedimetric immunosensor and redox couple ([(Fe(CN)6)]3-/4-) as a probe. The immunosensor was effectively developed and applied in the detection of SARS-CoV-2 spike protein RBD in saliva samples.

Reduced Graphene Oxide-Based Impedimetric Immunosensor for Detection of Enterotoxin A in Milk Samples.

A simple, cheap, and less aggressive immobilization procedure for biomolecules using reduced graphene oxide (rGO) was employed to prepare an impedimetric immunosensor for detection of staphylococcal enterotoxin A (SEA) from Staphylococcus aureus in milk samples. The scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS) were used to monitor the single steps of the electrode assembly process. The glassy carbon (GC)/rGO platform detected the antigen-antibody binding procedures of SEA with concentrations of 0.5 to 3.5 mg L-1 via impedance changes in a low frequency range. The impedimetric immunosensor was successfully applied for the determination of SEA in milk samples.

Risk of uterine cancer for BRCA1 and BRCA2 mutation carriers.

BACKGROUND: Whether BRCA1 and BRCA2 mutation carriers have a clinically relevant elevated risk of uterine cancer has implications for risk-reducing surgery. AIM: This multicentre, prospective cohort study assessed uterine cancer risk for mutation carriers compared with the general population. METHODS: Eligible mutation carriers were enrolled in the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) cohort study, had a uterus present and no history of uterine cancer at cohort entry. Epidemiological, lifestyle and clinical data were collected at cohort entry and updated three-yearly. Cancer events were verified using pathology reports. Follow-up was censored at death or last contact. Relative risk of uterine cancer was estimated using the standardised incidence ratio (SIR), with the expected number of cases determined using population-based data for Australia. RESULTS: Of 1,111 mutation carriers in kConFab, 283 were excluded due to prior hysterectomy (N = 278), prior uterine cancer (N = 2) or being non-residents (N = 3). After a median follow-up of 9.0 years, five incident uterine cancers were reported in the 828 eligible women (419 had prior breast cancer and 160 had prior tamoxifen use), compared to 2.04 expected (SIR = 2.45; 95% confidence interval [CI]: 0.80-5.72; P = 0.11). In 438 BRCA1 mutation carriers and 390 BRCA2 mutation carriers, three and two incident cases of uterine cancer were reported, respectively, compared to 1.04 expected (SIR = 2.87; 95% CI: 0.59-8.43; P = 0.18) and 0.99 expected (SIR = 2.01; 95% CI: 0.24-7.30; P = 0.52), respectively. All cases were endometrioid subtype, International Federation of Gynaecology and Obstetrics stage I-II disease. No serous uterine cancers were reported. CONCLUSIONS: Our findings are consistent with those from most other reports and do not support routine risk-reducing hysterectomy for BRCA1 and BRCA2 mutation carriers.

Effects of rapid temperature fluctuations prior to breeding on reproductive efficiency in replacement gilts.

Rapidly cooling pigs after heat stress (HS) results in a pathophysiological condition, and because rapid temperature fluctuations may be associated with reduced reproductive success in sows, it lends itself to the hypothesis that these conditions may be linked. Objectives were to determine the effects of rapid cooling on thermal response and future reproductive success in pigs. Thirty-six replacement gilts (137.8±0.9kg BW) were estrus synchronized and then 14.1±0.4 d after estrus confirmation, pigs were exposed to thermoneutral conditions (TN; n=12; 19.7±0.9°C) for 6h, or HS (36.3±0.5°C) for 3h, followed by 3h of rapid cooling (HSRC; n=12; immediate TN exposure and water dousing) or gradual cooling (HSGC; n=12; gradual decrease to TN conditions) repeated over 2 d. Vaginal (TV) and gastrointestinal tract temperatures (TGI) were obtained every 15min, and blood was collected on d 1 and d 2 during the HS and recovery periods at 180 and 60min, respectively. Pigs were bred 8.3±0.8 d after thermal treatments over 2 d. Reproductive tracts were collected and total fetus number and viability were recorded 28.0±0.8 d after insemination. HS increased TV and TGI (P=0.01; 0.98°C) in HSRC and HSGC compared to TN pigs. During recovery, TV was reduced from 15 to 105min (P=0.01; 0.33°C) in HSRC compared to HSGC pigs, but no overall differences in TGI were detected (P<0.05; 39.67°C). Rapid cooling increased (P<0.05) TNFα compared to HSGC and TN pigs during recovery-d 1 (55.2%), HS-d 2 (35.1%), and recovery-d 2 (64.9%). Viable fetuses tended to be reduced (P=0.08; 10.5%) and moribund fetuses tended to be increased (P=0.09; 159.3%) in HSRC compared to HSGC and TN pigs. In summary, rapid cooling prior to breeding may contribute to reduced fetal viability and reproductive success in pigs.

Hyperactivation of the Insulin Signaling Pathway Improves Intracellular Proteostasis by Coordinately Up-regulating the Proteostatic Machinery in Adipocytes.

Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy. Unexpectedly, this marked elevation in protein synthesis was accompanied by enhanced protein stability and folding and not by markers of proteostasis stress such as protein carbonylation and aggregation. The improvement in proteostasis was attributed to a coordinate up-regulation of proteins in the global proteostasis network, including ribosomal, proteasomal, chaperone, and endoplasmic reticulum/mitochondrial unfolded protein response proteins. We conclude that defects associated with hyperactivation of the insulin signaling pathway are unlikely attributed to defective proteostasis because up-regulation of protein synthesis by insulin is accompanied by up-regulation of proteostatic machinery.

Theme and variations: amphibious air-breathing intertidal fishes.

Over 70 species of intertidal fishes from 12 families breathe air while emerging from water. Amphibious intertidal fishes generally have no specialized air-breathing organ but rely on vascularized mucosae and cutaneous surfaces in air to exchange both oxygen and carbon dioxide. They differ from air-breathing freshwater fishes in morphology, physiology, ecology and behaviour. Air breathing and terrestrial activity are present to varying degrees in intertidal fish species, correlated with the tidal height of their habitat. The gradient of amphibious lifestyle includes passive remainers that stay in the intertidal zone as tides ebb, active emergers that deliberately leave water in response to poor aquatic conditions and highly mobile amphibious skipper fishes that may spend more time out of water than in it. Normal terrestrial activity is usually aerobic and metabolic rates in air and water are similar. Anaerobic metabolism may be employed during forced exercise or when exposed to aquatic hypoxia. Adaptations for amphibious life include reductions in gill surface area, increased reliance on the skin for respiration and ion exchange, high affinity of haemoglobin for oxygen and adjustments to ventilation and metabolism while in air. Intertidal fishes remain close to water and do not travel far terrestrially, and are unlikely to migrate or colonize new habitats at present, although in the past this may have happened. Many fish species spawn in the intertidal zone, including some that do not breathe air, as eggs and embryos that develop in the intertidal zone benefit from tidal air emergence. With air breathing, amphibious intertidal fishes survive in a variable habitat with minimal adjustments to existing structures. Closely related species in different microhabitats provide unique opportunities for comparative studies.

Brave new propagules: terrestrial embryos in anamniotic eggs.

A surprisingly large number of fish and amphibian species reproduce terrestrially despite the absence of the key evolutionary innovation of the amniotic egg. In contrast with shelled eggs of reptiles and birds, eggs of teleost fish and amphibians are typically much smaller and enclosed in relatively simple chorionic membranes. Incubation times may be brief or prolonged, and resultant hatchlings typically require the return to an aquatic habitat. Advantages of terrestrial incubation include the increased availability of warmer temperatures and avoidance of aquatic hypoxia, whereas disadvantages include desiccation, exposure to novel predators, and the risk of hatching into a hostile habitat. Hatching may be environmentally cued. Use of energy in the yolk may require trade-offs between growth of the embryo and extended incubation, as exemplified by a case study of the California Grunion. The physical challenges of terrestrial incubation, constraints for hatching, effects of egg size, and parental care are explored. Eight different types of early life history among anamniotic embryos incubating in a terrestrial environment are identified, with examples of these alternate routes to the invasion of land by vertebrates.

Mutant torsinA interacts with tyrosine hydroxylase in cultured cells.

A specific mutation (DeltaE302/303) in the torsinA gene underlies most cases of dominantly inherited early-onset torsion dystonia. This mutation causes the protein to aggregate and form intracellular inclusion bodies in cultured cells and animal models. Co-expression of the wildtype and mutant proteins resulted in the redistribution of the wildtype protein from the endoplasmic reticulum to inclusion bodies in cultured HEK293 cells, and this was associated with increased interaction between the two proteins. Expression of DeltaE302/303 but not wildtype torsinA in primary postnatal midbrain neurons resulted in the formation of intracellular inclusion bodies, predominantly in dopaminergic neurons. Tyrosine hydroxylase was sequestered in these inclusions and this process was mediated by increased protein-protein interaction between mutant torsinA and tyrosine hydroxylase. Analysis in an inducible neuroblastoma cell culture model demonstrated altered tyrosine hydroxylase activity in the presence of the mutant but not wildtype torsinA protein. Our results suggest that the interaction of tyrosine hydroxylase and mutant torsinA may contribute to the phenotype and reported dopaminergic dysfunction in torsinA-mediated dystonia.

Embryonic staging series for the beach spawning, terrestrially incubating California grunion Leuresthes tenuiswith comparisons to other Atherinomorpha.

California grunion Leuresthes tenuis synchronize spawning with tidal cycles, so the embryos incubate in a terrestrial environment, delay hatching until cued by a specific environmental trigger, and may extend incubation for up to an additional four weeks. These adaptations, however, do not appear to alter the morphology or sequence of early developmental stages as compared to other Atherinomorph fishes in the Orders Beloniformes and Cyprinodontiformes. Embryonic development is described in a series of 30 stages based on morphology observed by light microscopy. Stages are placed in five periods: zygote and cleavage, blastula, gastrula, segmentation and organogenesis, and hatching competence. Embryos from a southern population of L. tenuis in Los Angeles are compared with embryos found > 560 km north in San Francisco Bay. Northern L. tenuis embryos developed more slowly at several stages than southern embryos and reached hatching competence later, but both locations maintained synchrony with the tidal cycle for both spawning and hatching. The variation in rates of development and stage at hatching readiness are forms of developmental heterochrony that may be associated with evolutionary adaptation or morphological plasticity within this highly successful clade.

Interleukin-4 -590 (C>T), toll-like receptor-2 +2258 (G>A) and matrix metalloproteinase-9 -1562 (C>T) polymorphisms in pre-eclampsia.

Functional single nucleotide polymorphisms (SNPs) of interleukin (IL)-4 -590 (C>T), toll-like receptor (TLR)-2 +2258 (G>A) and matrix metalloproteinase (MMP)-9 -1562 (C>T) were examined by polymerase chain reaction-restriction fragment length polymorphism to identify their merit as genetic markers for pre-eclampsia. One hundred and seventeen pre-eclamptic women and 146 control subjects with uncomplicated singleton pregnancies participated in this study, conducted at Leeds General Infirmary and St James's University Hospital. While the TLR-2 +2258 (G>A) and MMP-9 -1562 (C>T) SNPs failed to present any significant association with pre-eclampsia, there was a marked trend for an association between the IL-4 -590 (C>T) SNP and pre-eclampsia (chi(2)= 5.87, P = 0.055), with a prevalence of TT homozygous women in this group (OR 4.455, 95% CI 1.286-15.350).

Impact of hyperglycemia on early embryo development and embryopathy: in vitro experiments using a mouse model.

BACKGROUND: The aim of this study is to model the processes of early embryopathy seen in human pregnancy complicated by maternal hyperglycemia secondary to maternal diabetes using a mouse embryo culture system. METHODS: Female mice were superovulated and mated in pairs. Two-cell embryos were harvested from the oviducts and cultured in vitro in KSOM medium (synthetic oviductal medium enriched with potassium) supplemented with 0.2, 5.56, 15.56 or 25.56 mM d-glucose. Cell proliferation, differentiation and apoptosis were assessed. Experiments were performed in constant, embryos exposed to a particular concentration of glucose (0.2, 5.56, 15.56 or 25.56 mM) from harvest to either Day 5 post fertilization (pf) or Day 8 pf, and fluctuating, embryos exposed to alternate high 25.56 mM and normal 5.56 mM concentrations of glucose between harvest and Day 5 pf, glycemic culture. RESULTS: Expected levels of blastocyst formation and hatching were seen at 0.2 and 5.56 mM concentrations of glucose but both were impaired at higher concentrations (chi(2), P < 0.005; P < 0.001). Total cell numbers (P < 0.002) and cell allocation to the inner cell mass (P < 0.01) were reduced, but with no evidence of enhanced apoptosis in the hyperglycemic cultures. Variation in hyperglycemic exposure of the embryos on Days 2, 3 and 4 showed no adverse effects of hyperglycemia up to 24 h, but 48 and 72 h exposures were equally embryopathic (P < 0.01). CONCLUSIONS: Hyperglycemic exposure for >24 h is toxic to early embryo development. These findings may explain the lower than expected implantation rates and higher than expected rates of congenital abnormality and early pregnancy loss seen in patients with diabetes, particularly those with poor diabetic control.

The myo-inositol-1-phosphate synthase gene is essential in Trypanosoma brucei.

The de novo synthesis of myo-inositol occurs via a two-step process: first, glucose 6-phosphate is converted into inositol 1-phosphate by an INO1 (myo-inositol-1-phosphate synthase; EC 5.5.1.4); then, it is dephosphorylated by an inositol monophosphatase. The myo-inositol can then be incorporated into PI (phosphatidylinositol), which is utilized in a variety of cellular functions, including the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. A putative INO1 was identified in the Trypanosoma brucei genome database and, by recombinant expression in Escherichia coli, was shown to be a catalytically active INO1. To investigate the importance of INO1, we created a conditional knockout, which, under non-permissive conditions, showed that INO1 is an essential gene in bloodstream form T. brucei and that the de novo synthesized myo-inositol is used for the formation of PI and GPI anchors.

Beach-spawning fishes, terrestrial eggs, and air breathing.

Many fishes have independently evolved beach spawning with oviposition at the water's edge. These include intertidal, subtidal, and estuarine, as well as a few freshwater, species. Their spectacular reproductive behavior at the boundary of water and land has focused attention on adults, but they emerge either briefly or not at all. The need for air breathing is more apparent in the eggs, and the reasons for emergence are more applicable to eggs than to the adults of most beach-spawning fishes. There is little evidence of air breathing in the adults, unless they are regularly emerged at other times as well. Conversely, eggs metabolize in air and show substantial emergence tolerance. We consider beach spawning a form of parental care in fishes. The adults place eggs so they will be emerged into air during part or all of incubation, providing increased temperatures, oxygen availability, and protection. Beach spawning provides habitat segregation at different points in the life history, with air emergence early in the life cycle and a return to water at hatching. The parents take great risks to spawn at the water's edge to give their offspring the most advantageous beginning in life.

Structure of epiglucan, a highly side-chain/branched (1 --> 3;1 --> 6)-beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht.

The extracellular fungal polysaccharide, epiglucan, synthesised by Epicoccum nigrum is a side-chain/branched (1 --> 3;1 --> 6)-D-beta-glucan. Methylation analysis, 13C DEPT NMR and specific enzymic digestion data show slight variation in branching frequency among the epiglucans from the three strains examined. The (1 --> 3)-beta-linked backbone has (1 --> 6)-beta-linked branches at frequencies greater than the homologous glucans, scleroglucan and schizophyllan, from Sclerotium spp. and Schizophyllum commune, respectively. The structural analyses do not allow a distinction to be made between structures I and II. [structures: see text] Epiglucan displays non-Newtonian shear thinning rheological properties, typical of these glucans.

The rate and anisotropy of impulse propagation in the postnatal terminal crest are correlated with remodeling of Cx43 gap junction pattern.

BACKGROUND: Disruptions to intermyocyte coupling have been implicated in arrhythmogenesis and development of conduction disturbances. At present, understanding of the relationship between the microscopic organization of intercellular coupling and the macroscopic spread of impulse in the normal and diseased heart is largely confined to theoretical analyses. METHODS AND RESULTS: The abundance and arrangement of gap junctions, as well as conduction properties, were assessed in terminal crest preparations isolated from the atria of neonate, weanling, and adult rabbits. We report that the connexin composition of terminal crest was uncomplicated, with Cx43 being the most prominent isoform detectable by Western blotting and immunostaining. Terminal crest myocytes showed little change in total Cx43-gap junction per cell during postnatal growth as assessed by stereology. However, marked non-uniformities emerged in the sarcolemmal distribution of Cx43-gap junctions. Cx43-gap junction area at myocyte termini increased 3.5-fold from birth to adulthood. Correlated with this change in Cx43, impulse propagation velocity parallel to the myofiber axis, as assessed by multi-site optical mapping using voltage-sensitive dye (di-4-ANEPPS), increased 2.4-fold. Conversely, the amount of Cx43-gap junctions on myocyte sides, and the conduction velocity transverse to the myofiber axis, remained relatively invariant during maturation. Hence, the increasing electrical anisotropy of maturing terminal crest was wholly accounted for by increases in conductance velocity along the bundle. This increase in longitudinal conduction velocity was correlated with changes in the sarcolemmal pattern, but not the overall density, of Cx43-gap junctions. CONCLUSIONS: This study provides the first correlative structure/function analysis of the relationship between the macroscopic conduction of impulse and the microscopic cellular organization of gap junctions in a differentiating cardiac bundle. Confirmation is provided for theoretical predictions which emphasize the importance of the cell-to-cell geometry of coupling in determining the spread and pattern of myocardial activation.

Role of developmental factors in the switch from pyruvate to glucose as the major exogenous energy substrate in the preimplantation mouse embryo.

Preimplantation mouse embryos, cultured in vitro and those freshly flushed from the reproductive tract, exhibit a switch in energy substrate preference, from pyruvate during the early preimplantation stages, to glucose at the blastocyst stage. Although the biochemical basis of this phenomenon is quite well characterized, its timing and possible association with developmental factors have not been considered. We have therefore examined the role of five developmental factors in determining the timing of the switch, namely: (1) embryo age (in hours post hCG); (2) developmental stage; (3) cytokinesis; (4) cell number; and (5) activation of the embryonic genome. One-cell embryos, which develop more slowly than 2-cell embryos in vitro, were used to investigate the role of embryo age and developmental stage. Cytochalasin D, which inhibits cytokinesis and delays the timing of compaction and cavitation, was used to investigate the role of cell division and developmental stage. Finally, transcription of the embryonic genome was examined with the inhibitor, alpha-amanitin. Pyruvate and glucose consumption by single embryos were measured using a noninvasive ultramicrofluorometric technique. The results showed that the timing of the switch in energy substrate preference is precisely regulated in the mouse preimplantation embryo. Activation of the embryonic genome is a prerequisite for the switch and its timing is closely associated with developmental stage, specifically compaction and/or cavitation. Cell number, cytokinesis and embryo age appeared to be unrelated to the timing of the switch. These conclusions may well be extrapolated to other species, since an increase in net glucose uptake, if not always at the expense of pyruvate, is a feature of preimplantation embryo metabolism in all mammals studied.

Heparin-binding epidermal growth factor significantly improves human blastocyst development and hatching in serum-free medium.

The purpose of this study was to determine the effect of heparin-binding epidermal growth factor (HB-EGF) on human embryo development in vitro from days 2 to 14 post-insemination. Embryos were cultured in a complex serum-free medium (CSFM3) in the absence and presence of 1 nM and 100 nM HB-EGF. Development to the blastocyst stage of A-C-grade embryos (A grade = highest quality) was improved in the presence of 1 nM HB-EGF from 40.7% to 65.4% and significantly increased to 71.0% in the presence of 100 nM HB-EGF (P < 0.05). Moreover, the percentage of blastocysts hatching was improved in the presence of 1 nM HB-EGF from 45.5% to 70.5% and almost doubled to 81.8% (P < 0.05) in the presence of 100 nM HB-EGF. HB-EGF promoted the development of high-grade (classed as BG1) and medium-grade (BG2) blastocysts. There was no difference in blastocyst quality between the control and HB-EGF-treated embryos as assessed by blastocyst cell number and consumption of the major energy substrates, pyruvate and glucose, measured on day 6 of culture. Further development was assessed by culturing the blastocysts on growth factor-reduced Matrigel (GFR-Matrigel). Adherence and outgrowth were observed, with these embryos producing significantly more human chorionic gonadotrophin over days 7-14 compared with those cultured on plastic (47.8 +/- 8.0 mU versus 23.0 +/- 8.6 mU). The addition of recombinant human growth factors to clinical in-vitro fertilization medium may be useful in promoting embryo development with a view to carrying out blastocyst transfers.