Emerging Trends in Cryo-EM-based Structural Studies of Neuropathological Amyloids.

Tauopathies, synucleinopathies, Aß amyloidosis, TDP-43 proteinopathies, and prion diseases- these neurodegenerative diseases have in common the formation of amyloid filaments rich in cross-ß sheets. Cryo-electron microscopy now permits the visualization of amyloid assemblies at atomic resolution, ushering a wide range of structural studies on several of these poorly understood amyloidogenic proteins. Amyloids are polymorphic with minor modulations in reaction environment affecting the overall architecture of their assembly, making amyloids an extremely challenging venture for structure-based therapeutic intervention. In 2017, the first cryo-EM structure of tau filaments from an Alzheimer's disease-affected brain established that in vitro assemblies might not necessarily reflect the native amyloid fold. Since then, brain-derived amyloid structures for several proteins across many neurodegenerative diseases have uncovered the disease-relevant amyloid folds. It has now been shown for tauopathies, synucleinopathies and TDP-43 proteinopathies, that distinct amyloid folds of the same protein might be related to different diseases. Salient features of each of these brain-derived folds are discussed in detail. It was also recently observed that seeded aggregation does not necessarily replicate the brain-derived structural fold. Owing to high throughput structure determination, some of these native amyloid folds have also been successfully replicated in vitro. In vitro replication of disease-relevant filaments will aid development of imaging ligands and defibrillating drugs. Towards this direction, recent high-resolution structures of tau filaments with positron emission tomography tracers and a defibrillating drug are also discussed. This review summarizes and celebrates the recent advancements in structural understanding of neuropathological amyloid filaments using cryo-EM.

Highly pathogenic avian influenza (H5N1) infection in crows through ingestion of infected crow carcasses.

The present study was aimed to investigate the role of cannibalism in transmission of H5N1 avian influenza virus to house crows (Corvus splendens). Four crows were intranasally inoculated with 108.0 EID50 (A/crow/India/01CA249/2021) H5N1 highly pathogenic avian influenza (HPAI) virus and were observed for 14 days for any overt signs of illness. Two of the infected crows showed signs of wing paralysis, incoordination, and torticollis. For cannibalism experiment, two crows showing clinical signs were euthanized on 14th day post-infection (dpi) and were kept in the isolator and four naïve healthy crows were introduced along with the euthanized crows. The viscera from the infected carcasses were eaten by all the four crows. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. All four crows showed clinical signs viz., dullness, reluctance to move with ruffled feathers on 6th day post cannibalism along with neurological signs including incoordination and paralysis of the wings. All the crows gradually recovered after showing clinical signs and were euthanized on 21st day of observation period. Virus excretion was observed from 3rd to 11th day post cannibalism through both oropharyngeal and cloacal routes with maximum shedding through oropharyngeal route. The virus was isolated from lungs and trachea of one the infected crows at 21st day after euthanasia. All the four crows seroconverted against H5N1 virus infection at 14th day post cannibalism. Our study confirms the transmission of H5N1 virus in crows through cannibalism and highlights how H5N1 virus might circulate in a crow colony once they become infected.

Food Order and Timing Effects on Glycaemic and Satiety Responses to Partial Fruit-for-Cereal Carbohydrate Exchange: A Randomized Cross-Over Human Intervention Study.

Postprandial glycaemic response amplitude plays a critical role in diabetic complications, but is subject to food order and temporal separation within a meal. Effects of partial fruit-for-cereal carbohydrate exchange on glycaemic and appetite responses, as affected by food order and separation, were examined using kiwifruit (KF) and wheaten breakfast cereal biscuit (WB). In a randomized cross-over intervention study, 20 subjects ingested 51.7 g of available carbohydrate as 74 g WB alone, or as 200 g KF and 37 g WB, each delivering 25.85 g of available carbohydrate. The 200 g KF was partially exchanged for 37 g of WB, at 90 min and 30 min before, at the same time as, or 30 min after, ingesting WB. Incremental satiety responses were derived from appetite scores measured using a visual analogue scale, and capillary blood glucose responses were monitored. In all exchanges, KF reduced the glycaemic response (iAUC) by 20-30% with no loss of total satiation. The incremental glycaemic and satiety responses to food ingestion followed each other closely. Glycaemic response amplitudes were reduced almost 50% compared with 74 g WB when KF ingestion preceded WB ingestion by 30 min, and less when the KF was ingested with or 30 min after the cereal. The results suggest that fruit most effectively suppresses the digestion of cereal carbohydrates if ingested long enough before the cereal to prevent overlap of the glycaemic responses, but close enough for fruit components that impede carbohydrate digestion or uptake to interact with the ingested cereal in the gut. Ethics approval was obtained from the Human and Disabilities Ethics Committee (HDEC) of the New Zealand Ministry of Health. The trial was registered with the Australian New Zealand Clinical Trials Registry (Trial ID: ACTRN12615000744550).

Cryo-EM-based structural insights into supramolecular assemblies of γ-hemolysin from S. aureus reveal the pore formation mechanism.

γ-Hemolysin (γ-HL) is a hemolytic and leukotoxic bicomponent ß-pore-forming toxin (ß-PFT), a potent virulence factor from the Staphylococcus aureus Newman strain. In this study, we performed single-particle cryoelectron microscopy (cryo-EM) of γ-HL in a lipid environment. We observed clustering and square lattice packing of octameric HlgAB pores on the membrane bilayer and an octahedral superassembly of octameric pore complexes that we resolved at resolution of 3.5 Å. Our atomic model further demonstrated the key residues involved in hydrophobic zipping between the rim domains of adjacent octameric complexes, providing additional structural stability in PFTs post oligomerization. We also observed extra densities at the octahedral and octameric interfaces, providing insights into the plausible lipid-binding residues involved for HlgA and HlgB components. Furthermore, the hitherto elusive N-terminal region of HlgA was also resolved in our cryo-EM map, and an overall mechanism of pore formation for bicomponent ß-PFTs is proposed.

A Study of Arginase Expression in Chronic Non-healing Wounds.

Arginase expression has been recently shown to increase in numerous disease states like neurodegeneration, inflammation, and malignancies. Although it has been found to be functionally important in various disease pathologies, little is known about its role in wound healing. Here, we look at the expression of arginase and its isoforms in chronic non-healing wounds and also study the expression of nitric oxide synthase (NOS) and oxidative stress enzymes in them. Wound tissues and blood samples were collected at the time of index presentation and follow-up from 61 chronic non-healing wound cases. The expression patterns of arginase isoenzymes, NOS, superoxide dismutases (SOD), lactic acid dehydrogenase (LDH), and catalase were examined by using enzyme-linked immunosorbent assay, immunohistochemistry, and western blot analysis at the transcript and protein level. We reported a significant decrease of serum arginase levels in chronic nonhealing wounds in the progress of wound healing. Interestingly, tissue arginase levels were found to be increased with improved wound condition at follow-up. Tissue NOS, LDH, and catalase activity were also found to be increased with the progress of healing, whereas SOD levels were downregulated. Our findings reported increased expression at the transcript level of arginase-I and arginase-II in chronic non-healing wounds for the first time. In conclusion, we observed decreased serum arginase levels in completely healed patients as compared to non-healed cases. Our study findings support the hypothesis that inhibition of the activity of arginase delays wound healing. Arginase and iNOS may also find their place in the future as possible biomarkers for wound healing.

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM.

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)-the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.

Effects of Daily Ingestion of Two SunGold Kiwifruit for 6 Weeks on Metabolic and Inflammatory Biomarkers: A Randomized, Cross-Over, Exploratory Intervention Study.

Kiwifruit contain many components, some considered beneficial, such as vitamins, phytochemicals and dietary fibre, and others potentially harmful, such as fructose and glucose in fruit sugars. In a 6-week, randomised, crossover study aimed at exploring the net effects of daily consumption of kiwifruit, 23 healthy participants consumed two Actinidia chinensis var. chinensis 'Zesy002' (marketed as Zespri™ SunGold™ Kiwifruit) per day as part of their customary diet (intervention) or without kiwifruit (control) as their customary diet for 6 weeks in a cross-over study. Anthropometric data, venous blood, and urine samples were collected at the start and end of the 6-week intervention and control periods for the measurement of physical changes, plasma glucose, insulin, glycated haemoglobin, short-chain fatty acids, blood lipids, uric acid, inflammatory biomarkers, and urinary ascorbic acid. Variables were measured between the start and finish of interventions, and between intervention and control periods. Food diaries were completed on the 3 days before blood sampling to estimate dietary ascorbic acid and dietary fibre intakes. Despite urinary vitamin C and food diaries indicating compliance, and good precision in measurements, there were no appreciable changes in biomarkers during the study, either within or between intervention and control periods, that would indicate a change in health status. Thus, the sizes of any effects of kiwifruit ingestion were too small to become significant under the test conditions used, indicating a high probability that daily ingestion of two SunGold kiwifruit is safe with respect to metabolic health.

Analysis of cellular and cell free mitochondrial DNA content and reactive oxygen species levels in maternal blood during normal pregnancy: a pilot study.

BACKGROUND: Alterations in mitochondrial signatures such as mitochondrial DNA (mtDNA) content in maternal blood have been linked to pregnancy-related complications. However, changes in maternal mtDNA content, their distribution and associated signaling during normal pregnancies are not clear; which could suggest their physiological role in maternal adaptation to pregnancy related changes and a reference threshold. THE AIM OF THIS STUDY: to assess the distribution of mtDNA in peripheral blood and their association with circulatory ROS levels across different trimesters of healthy pregnancy. METHODS: In this pilot cross sectional study, blood samples of normal pregnant women from each trimester (total = 60) and age-matched non-pregnant (NP) women as control group (n = 20) were analyzed for a) the relative distribution of mtDNA content in cellular and cell free (plasma) fractions using relative quantitative polymerase chain reaction (qPCR) and b) the levels of circulating reactive oxygen species (ROS) by measurement of plasma H2O2. The results were compared between pregnant and NP groups and within trimesters for significant differences, and were also analyzed for their correlation between groups using statistical methods. RESULTS: While, we observed a significant decline in cellular mtDNA; plasma mtDNA was significant increased across all trimesters compared to NP. However, from comparisons within trimesters; only cellular mtDNA content in 3rd trimester was significantly reduced compared to 1st trimester, and plasma mtDNA did not differ significantly among different trimesters. A significantly higher level of plasma H2O2 was also observed during 3rd trimester compared to NP and to 1st trimester. Correlation analysis showed that, while cellular mtDNA content was negatively correlated to plasma mtDNA and to plasma H2O2 levels; plasma mtDNA was positively correlated with plasma H2O2 content. CONCLUSIONS: This study suggested that normal pregnancy is associated with an opposing trend of reduced cellular mtDNA with increased circulatory mtDNA and H2O2 levels, which may contribute to maternal adaptation, required during different stages of pregnancy. Estimation of mtDNA distribution and ROS level in maternal blood could show mitochondrial functionality during normal pregnancy, and could be exploited to identify their prognostic/ diagnostic potential in pregnancy complications.

Phage-tail-like bacteriocins as a biomedical platform to counter anti-microbial resistant pathogens.

Phage Tail Like bacteriocins (PTLBs) has been an area of interest in the last couple of years owing to their varied application against multi-drug resistant (MDR), anti-microbial resistant (AMR) pathogens and their evolutionary link with the dsDNA virus and bacteriophages. PTLBs are defective phages derived from Myoviridae and Siphoviridae phages, PTLBs are distinguished into R-type (Rigid type) characterized by a non-flexible contractile nanotube resembling Myoviridae phage contractile tails, and F-type (Flexible type) with a flexible non-contractile rod-like structure similar to Siphoviridae phages. In this review, we have discussed the structural association, mechanism, and characterization of PTLBs. Moreover, we have elucidated the symbiotic biological function and application of PTLBs against MDR and XDR pathogens and highlighted the evolutionary role of PTLBs. The difficulties that must be overcome to implement PTLBs clinically are also discussed. It is imperative that these issues be addressed by academics in future studies before being implemented in clinical settings. This article is novel in its way as it will not only provide us with a gateway that acts as a novel strategy for scholars to mitigate and control the uprising issue of AMR pathogens but also promote the development of clinical studies for PTLBs.

Dietary Fibre and Organic Acids in Kiwifruit Suppress Glycaemic Response Equally by Delaying Absorption-A Randomised Crossover Human Trial with Parallel Analysis of 13C-Acetate Uptake.

Non-sugar components of kiwifruit reduce the amplitude of the glycaemic response to co-consumed cereal starch. We determined the relative contribution of different non-sugar kiwifruit components to this anti-glycaemic effect. Healthy participants (n = 9) ingested equal carbohydrate meals containing 20 g starch as wheat biscuit (WB, 30 g), and the sugar equivalent of two kiwifruit (KFsug, 20.4 g), either intrinsic or added as glucose, fructose and sucrose (2:2:1). The meals were WB+KFsug (control, no non-sugar kiwifruit components), WB + whole kiwifruit pulp (WB+KF), WB + neutralised kiwifruit pulp (WB+KFneut), WB + low-fibre kiwifruit juice (WB+KFjuice) and WB+KFsug + kiwifruit organic acids (WB+KFsug+OA). All meals were spiked with 100 mg sodium [1-13C] acetate to measure intestinal absorption. Each participant ingested all meals in random order. Blood glucose and breath 13CO2 were measured at ingestion and at 15 min intervals up to 180 min. Compared with WB+KFsug, whole kiwifruit pulp (WB+KF) almost halved glycaemic response amplitude (p < 0.001), reduced incremental area under the blood glucose response curve (iAUC) at 30 min (peak) by 50% (p < 0.001), and averted late postprandial hypoglycaemia. All other treatments suppressed response amplitude half as much as whole kiwifruit and averted acute hypoglycaemia, with little effect on iAUC. Effects on 13CO2 exhalation paralleled effects on blood glucose (R2 = 0.97). Dietary fibre and organic acids contributed equally to the anti-glycaemic effect of kiwifruit by reducing intestinal absorption rate. Kiwifruit flesh effectively attenuates glycaemic response in carbohydrate exchange, as it contains fructose, dietary fibre and organic acids.

Chewing differences in consumers affect the digestion and colonic fermentation outcomes: in vitro studies.

It is important to understand variability in consumer chewing behavior for designing food products that deliver desired functionalities for target consumer segments. In this study, we selected 29 participants, representing the large range of chewing variation we had observed in 142 healthy young adults, and investigated the influence of chewing behavior on gastrointestinal digestion and colonic fermentation, using in vitro models and brown rice as a model food. Chewing behavior measured by video observations and chewing outcome differed widely between participants, resulting in large differences in the digestibility of carbohydrates. Inter-individual differences in chewing behavior and chewing outcome also significantly affected in vitro patterns of microbial composition and the production of organic acid metabolites, resulting from colonic fermentation, which is increasingly recognized to be important for human health. These digestion/fermentation outcomes were largely related with the chewing time per mouthful, proportion of bolus particles bigger than 2 mm and amount of saliva added to the bolus during chewing. No significant relationships were found with other chewing trajectory and oral physiological measures. These results suggest that modification of chewing may be an effective strategy to control blood glucose levels and to shape gut microbiota and their metabolites, without altering diets, and that further in vivo studies are warranted to confirm these in vitro findings.

Metabolic and Blood Pressure Effects of Consuming Two Kiwifruit Daily for 7 Weeks: A Randomised Controlled Trial.

Background: Eating two kiwifruit before breakfast by equi-carbohydrate partial exchange of cereal has been associated with lower postprandial glucose and insulin, but it increases the intake of fruit sugar. We assessed the effects of kiwifruit ingestion at breakfast over 7 weeks on metabolic and physiologic factors. Method: Forty-three healthy Asian participants were randomised to ingest 500 mL of carbonated water (control) or 500 mL of carbonated water plus two kiwifruit (intervention), before breakfast. Three-day weighed diet records were taken before and at week 4 during the intervention. Overnight fasting blood samples were taken at baseline and week 7. Forty-two participants completed the study (n = 22 control, n = 20 intervention). Results: The kiwifruit group consumed more fructose, vitamin C, vitamin E, and carbohydrates as a percentage of energy compared with the control group (p < 0.01). There was no evidence of between-group changes in metabolic outcomes at the end of the intervention, with the following mean (95% confidence interval) differences in fasting blood samples: glucose 0.09 (−0.06, 0.24) mmol/L; insulin −1.6 (−3.5, 0.3) µU/mL; uric acid −13 (−30, 4) µmol/L; triglycerides −0.10 (−0.22, 0.03) mmol/L; and total cholesterol −0.05 (−0.24, 0.14) mmol/L. There was a −2.7 (−5.5, 0.0) mmHg difference in systolic blood pressure for the intervention group compared with the control group. Conclusion: Eating two kiwifruit as part of breakfast increased fruit consumption and intake of antioxidant nutrients without a change in fasting insulin. There was a difference in systolic blood pressure and no adverse fructose-associated increases in uric acid, triglycerides, or total cholesterol. This simple intervention may provide health benefits to other demographic groups.

In Vitro Digestive Analysis of Digestible and Resistant Starch Fractions, with Concurrent Glycemic Index Determination, in Whole Grain Wheat Products Minimally Processed for Reduced Glycaemic Impact.

Eight wheat products differing in texture (porridge vs. bread), grain fineness (fine, kibbled, intact), and cooking (raw vs. cooked), with pre-measured glycaemic indexes (GI), were analysed by in vitro amylolytic digestion to determine effects of processing to reduce GI on quantities of starch fractions differing in digestibility. The accuracy and precision of the in vitro analysis was assessed from its ability to concurrently predict clinical GI. In porridges, kernel intactness and lack of cooking reduced GI while increasing Type 1 (inaccessible) and Type 2 (ungelatinised) resistant starch. Porridge in vitro GI values (GIiv), calculated from the area under in vitro digestion curves minus estimated blood glucose disposal, were: raw fine, 26.3; raw kibbled, 12.6; cooked fine, 63.9; cooked kibbled, 44.1; and correlated closely with clinical GI values (R2 = 0.97). In bread, the negative association of kernel intactness and resistant starch with GI was seen in vitro but not in vivo. Bread GIiv values were: roller milled flour, 67.4; stoneground flour 61.1; kibbled grain, 53.0; kibbled + intact kernel, 49.5; but correlation with clinical values was low (R2 = 0.47), and variability in the clinical results was high (clinical CV = 72.5%, in vitro CV = 3.7%). Low glycaemic potency of wheat by minimal processing was achieved by maintaining particle size, avoiding hydrothermal treatment, avoiding crushing and using a food matrix requiring little chewing for ingestion. Use of in vitro digestive analysis for high precision measurement of starch fractions with potential secondary health benefits was validated by accurate concurrent prediction of the glycaemic index but needed to account for effects of chewing.

Lactobacillus rhamnosus GG Evaluation in Acute Diarrhea (LEAD): An Observational Study.

Background and objective Probiotics with documented efficacy such as Lactobacillus rhamnosus GG(LGG) and Saccharomyces boulardii might be utilized as adjuncts to rehydration for the management of acute gastroenteritis (AGE) in children. In this study, we aimed to evaluate the potential role of LGG in acute diarrhea in the Indian pediatric population. Methods An observational, cross-sectional study [Lactobacillus rhamnosus GG Evaluation in Acute Diarrhea (LEAD)] was conducted among children aged one month to 12 years with acute watery diarrhea. In addition to standard management of diarrhea, LGG was given as an adjuvant treatment at the discretion of treating physicians based on their routine practice. Observations were documented on days one, three, and five. Outcomes such as frequency and duration of diarrhea, time to change in consistency of stools, Bristol Stool Chart (BSC) reading, and global assessment by healthcare practitioners (HCPs) and patients were also recorded. Results Of the 2,080 patients enrolled, 1,900 completed the five-day follow-up. There was marked improvement observed in the number of incidences of loose stools from day one (mean: 7.33) to day five (mean: 1.6). The mean time to improvement in stool consistency was 34 hours. The mean duration of diarrhea was 44.63 hours. A relatively shorter duration of diarrhea was reported among participants in this study. There was also a significant improvement in the number of vomiting episodes. Most patients and HCPs reported the product to be excellent/very good on the global assessment scale. No adverse effects were noted in any of the groups. Conclusion Based on our findings, LGG supplementation may be a beneficial adjuvant treatment in reducing the severity and duration of acute diarrheal episodes.

Effects of Xanthan Gum, Lambda-Carrageenan and Psyllium Husk on the Physical Characteristics and Glycaemic Potency of White Bread.

White bread contains a high proportion of easily digestible starch, which contributes to an undesirable rapid increase in blood glucose concentration. This study investigated the effects of nonstarch polysaccharides (NSP) -xanthan gum, lambda-carrageenan and psyllium husk on the physical functionality and glycaemic potency of white bread. The amount of water for each formulation was adjusted based on DoughLab set at a target torque value of ~500 FU for sufficient dough development. Adding NSP generally resulted in significantly increased loaf volumes and decreased hardness. The glycaemic potency (glycaemic glucose equivalents (GGE) g) of bread was found to be reduced with the addition of NSP at all levels (1, 3 and 5% w/w based on flour weight). Increasing the concentration of xanthan gum and lambda-carrageenan did not show any further decrease in the glycaemic potency. Notably, adding 5% w/w psyllium husk significantly reduced the glycaemic potency from ~49 GGE/100 g in the reference bread to 32 GGE/100 g. The reduction in the glycaemic potency was attributed to viscosity effects (for xanthan) and starch-NSP interactions (for psyllium husk). Overall, the 5% w/w psyllium husk bread sample was most promising in terms of both physical characteristics and its effect on in vitro glucose release.

Experimental Infection and In-Contact Transmission of H9N2 Avian Influenza Virus in Crows.

This study aimed to investigate the potential of H9N2 avian influenza virus to cause disease and intra-species transmission in house crows (Corvus splendens). A group of six crows were intranasally inoculated with 106.0 EID50 of H9N2 virus (A/chicken/India/07OR17/2021), and 24 h post-inoculation six naïve crows were co-housed with infected crows. Crows were observed for 14 days for any overt signs of illness. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. No apparent clinical signs were observed in either infected or in-contact crows. Virus excretion was observed only in infected birds up to 9 days post-infection (dpi) through both oropharyngeal and cloacal routes. All six infected crows seroconverted to H9N2 virus at 14 dpi, whereas all in-contact crows remained negative to H9N2 virus antibodies. No virus could be isolated from tissues viz., lung, liver, kidney, pancreas, small intestine and large intestine. Although crows became infected with the H9N2 virus, transmission of the virus was inefficient to the in-contact group. However, virus excretion through oral and cloacal swabs from infected crows suggests a potential threat for inter-species transmission, including humans. Crows, being a common synanthrope species, might have some role in influenza virus transmission to poultry and humans, which needs to be explored further.

Dynamical modeling of miR-34a, miR-449a, and miR-16 reveals numerous DDR signaling pathways regulating senescence, autophagy, and apoptosis in HeLa cells.

Transfection of tumor suppressor miRNAs such as miR-34a, miR-449a, and miR-16 with DNA damage can regulate apoptosis and senescence in cancer cells. miR-16 has been shown to influence autophagy in cervical cancer. However, the function of miR-34a and miR-449a in autophagy remains unknown. The functional and persistent G1/S checkpoint signaling pathways in HeLa cells via these three miRNAs, either synergistically or separately, remain a mystery. As a result, we present a synthetic Boolean network of the functional G1/S checkpoint regulation, illustrating the regulatory effects of these three miRNAs. To our knowledge, this is the first synthetic Boolean network that demonstrates the advanced role of these miRNAs in cervical cancer signaling pathways reliant on or independent of p53, such as MAPK or AMPK. We compared our estimated probability to the experimental data and found reasonable agreement. Our findings indicate that miR-34a or miR-16 may control senescence, autophagy, apoptosis, and the functional G1/S checkpoint. Additionally, miR-449a can regulate just senescence and apoptosis on an individual basis. MiR-449a can coordinate autophagy in HeLa cells in a synergistic manner with miR-16 and/or miR-34a.

Development of RNA-Based Assay for Rapid Detection of SARS-CoV-2 in Clinical Samples.

INTRODUCTION: The ongoing spread of pandemic coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of growing concern. Rapid diagnosis and management of SARS-CoV-2 are crucial for controlling the outbreak in the community. Here, we report the development of a first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 min. METHOD: We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and change in gold-colloid color from pink to blue visibility range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus-infected women. RESULTS: The results were validated using nasopharyngeal RNA samples from positive COVID-19 subjects (n = 136). Using real-time polymerase chain reaction as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. CONCLUSION: We found that the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

Glycaemic potency reduction by coarse grain structure in breads is largely eliminated during normal ingestion.

The hypothesis that coarse grain particles in breads reduce glycaemic response only if the particles remain intact during ingestion was tested. Three breads were formulated: (1) White bread (WB - reference), (2) 75 % of kibbled purple wheat in 25 % white bread matrix (PB) and (3) a 1:1 mixture of 37·5 % kibbled soya beans and 37·5 % of kibble purple wheat in 25 % white bread matrix (SPB). Each bread was ingested in three forms: unchewed (U), as customarily consumed (C) and homogenised (H). Twelve participants ingested 40 g available carbohydrate portions of each bread in each form, with post-prandial blood glucose measured over 120 min. Glycaemic responses to WB were the same regardless of its form when ingested. Unchewed PB had significantly less glycaemic effect than WB, whereas the C and H forms were similar to WB. Based on a glycaemic index (GI) of 70 for WB, the GI values for the C, U and H breads, respectively, were WB: 70·0, 70 and 70, PB: 75, 42 and 61, SPB: 57, 48 and 55 (%) (Least significant difference = 17·43, P < 0·05, bold numbers significantly different from WB). The similar glycaemic response to the H and C forms of the breads, and their difference from the U form, showed that the glycaemia-moderating effect of grain structure on starch digestion was lost during customary ingestion of bread. We conclude that the kibbled-grain structure may not effectively retard starch digestion in breads as normally consumed because it is largely eliminated by ingestive processes including chewing.

Exosomes Secreted by Umbilical Cord Blood-Derived Mesenchymal Stem Cell Attenuate Diabetes in Mice.

Mesenchymal stem cell (MSC) therapy is an innovative approach in diabetes due to its capacity to modulate tissue microenvironment and regeneration of glucose-responsive insulin-producing cells. In this study, we investigated the role of MSC-derived exosomes in pancreatic regeneration and insulin secretion in mice with streptozotocin-induced diabetes. Mesenchymal stem cells (MSCs) were isolated and characterized from umbilical cord blood (UCB). Exosomes were isolated and characterized from these MSCs. Diabetes was induced in male C57Bl/6 mice by streptozotocin (STZ; 40 mg/kg body weight, i.p.) for five consecutive days. The diabetic mice were administered (i.v.) with MSC (1 × 105 umbilical cord blood MSC cells/mice/day), their derived exosomes (the MSC-Exo group that received exosomes derived from 1 × 105 MSC cells/mice/day), or the same volume of PBS. Before administration, the potency of MSCs and their exosomes was evaluated in vitro by T cell activation experiments. After day 7 of the treatments, blood samples and pancreatic tissues were collected. Histochemistry was performed to check cellular architecture and ß cell regeneration. In body weight, blood glucose level, and insulin level, cell proliferation assay was done to confirm regeneration of cells after MSC and MSC-Exo treatments. Hyperglycemia was also attenuated in these mice with a concomitant increase in insulin production and an improved histological structure compared to mice in the PBS-treated group. We found increased expression of genes associated with tissue regeneration pathways, including Reg2, Reg3, and Amy2b in the pancreatic tissue of mice treated with MSC or MSC-Exo relative to PBS-treated mice. MicroRNA profiling of MSC-derived exosomes showed the presence of miRs that may facilitate pancreatic regeneration by regulating the Extl3-Reg-cyclinD1 pathway. These results demonstrate a potential therapeutic role of umbilical cord blood MSC-derived exosomes in attenuating insulin deficiency by activating pancreatic islets' regenerative abilities.