Exploring Three PIPs and Three TIPs of Grapevine for Transport of Water and Atypical Substrates through Heterologous Expression in aqy-null Yeast

Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (P_f) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl₂, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl₂. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO₂, H₂O₂ and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H₂O₂, suggesting the contribution of Vitis aquaporins in the transport of these species.     

Design of High Speed and Low Offset Dynamic Latch Comparator in 0.18 μm CMOS Process

The cross-coupled circuit mechanism based dynamic latch comparator is presented in this research. The comparator is designed using differential input stages with regenerative S-R latch to achieve lower offset, lower power, higher speed and higher resolution. In order to decrease circuit complexity, a comparator should maintain power, speed, resolution and offset-voltage properly. Simulations show that this novel dynamic latch comparator designed in 0.18 µm CMOS technology achieves 3.44 mV resolution with 8 bit precision at a frequency of 50 MHz while dissipating 158.5 µW from 1.8 V supply and 88.05 µA average current. Moreover, the proposed design propagates as fast as 4.2 nS with energy efficiency of 0.7 fJ/conversion-step. Additionally, the core circuit layout only occupies 0.008 mm².     

Genome‐wide association study identifies variation of glucosidase being linked to natural variation of the maximal quantum yield of photosystem II

The maximum quantum yield of photosystem II (as reflected by variable to maximum chlorophyll a fluorescence, F_v/F_m) is regarded as one of the most important photosynthetic parameters. The genetic basis underlying natural variation in F_v/F_m, which shows low level of variations in plants under non‐stress conditions, is not easy to be exploited using the conventional gene cloning approaches. Thus, in order to answer this question, we have followed another strategy: we used genome‐wide association study (GWAS) and transgenic analysis in a rice mini‐core collection. We report here that four single‐nucleotide polymorphisms, located in the promoter region of β‐glucosidase 5 (BGlu‐5), are associated with observed variation in F_v/F_m. Indeed, our transgenic analysis showed a good correlation between BGlu‐5 and F_v/F_m. Thus, our work demonstrates the feasibility of using GWAS to study natural variation in F_v/F_m, suggesting that cis‐element polymorphism, affecting the BGlu‐5 expression level, may, indirectly, contribute to F_v/F_m variation in rice through the gibberellin signaling pathway. Further research is needed to understand the mechanism of our novel observation.     

Feasibility and Acceptability of Global Positioning System (GPS) Methods to Study the Spatial Contexts of Substance Use and Sexual Risk Behaviors among Young Men Who Have Sex with Men in New York City: A P18 Cohort Sub-Study

Background

No global positioning system (GPS) technology study has been conducted among a sample of young gay, bisexual, and other men who have sex with men (YMSM). As such, the purpose of this study was to evaluate the feasibility and acceptability of using GPS methods to understand the spatial context of substance use and sexual risk behaviors among a sample of YMSM in New York City, a high-risk population.

Methods

Data came from a subsample of the ongoing P18 Cohort Study (n = 75). GPS feasibility and acceptability among participants was measured with: 1) a pre- and post-survey and 2) adherence to the GPS protocol which included returning the GPS device, self-report of charging and carrying the GPS device as well as objective data analyzed from the GPS devices. Analyses of the feasibility surveys were treated as repeated measures as each participant had a pre- and post-feasibility survey. When comparing the similar GPS survey items asked at baseline and at follow-up, we present percentages and associated p-values based on chi-square statistics.

Results

Participants reported high ratings of pre-GPS acceptability, ease of use, and low levels of wear-related concerns in addition to few concerns related to safety, loss, or appearance, which were maintained after baseline GPS feasibility data collection. The GPS return rate was 100%. Most participants charged and carried the GPS device on most days. Of the total of 75 participants with GPS data, 75 (100%) have at least one hour of GPS data for one day and 63 (84%) had at least one hour on all 7 days.

Conclusions

Results from this pilot study demonstrate that utilizing GPS methods among YMSM is feasible and acceptable. GPS devices may be used in spatial epidemiology research in YMSM populations to understand place-based determinants of health such as substance use and sexual risk behaviors.     

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.     

Exploring human-animal host interactions and emergence of COVID-19: Evolutionary and ecological dynamics

The novel coronavirus disease (COVID-19) that emerged in December 2019 had caused substantial morbidity and mortality at the global level within few months. It affected economies, stopped travel, and isolated individuals and populations around the world. Wildlife, especially bats, serve as reservoirs of coronaviruses from which the variant Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) emerged that causes COVID-19. In this review, we describe the current knowledge on COVID-19 and the significance of wildlife hosts in its emergence. Mammalian and avian coronaviruses have diverse host ranges with distinct lineages of coronaviruses. Recombination and reassortments occur more frequently in mixed-animal markets where diverse viral genotypes intermingle. Human coronaviruses have evolved through gene gains and losses primarily in interfaces where wildlife and humans come in frequent contact. There is a gap in our understanding of bats as reservoirs of coronaviruses and there is a misconception that bats periodically transmit coronaviruses to humans. Future research should investigate bat viral diversity and loads at interfaces between humans and bats. Furthermore, there is an urgent need to evaluate viral strains circulating in mixed animal markets, where the coronaviruses circulated before becoming adapted to humans. We propose and discuss a management intervention plan for COVID-19 and raise questions on the suitability of current containment plans. We anticipate that more virulent coronaviruses could emerge unless proper measures are taken to limit interactions between diverse wildlife and humans in wild animal markets.     

Kinetics and thermodynamics of a novel endoglucanase (CMCase) from <Emphasis Type="Italic">Gymnoascella </Emphasis><Emphasis Type="Italic">citrina</Emphasis> produced under solid-state condition

Gymnoascella citrina produced two isoforms of endoglucanases (CMCase-I and -capital I, Ukrainiancapital I, Ukrainian) under solid-state condition. Purified CMCase-I was novel because it was apparently holoenzyme in nature. The enzyme was monomeric as its native and subunit mass were almost the same, i.e., 43 and 42 kDa, respectively. Ea for carboxymethylcellulose (CMC) hydrolysis was 36.2 kJ mol(-1). The enzyme was stable over a pH range of 3.5-6.5, while temperature optimum was 55 degrees C. Vmax, Km and k (cat )for CMC hydrolysis were 39 U mg(-1) protein, 6.25 mg CMC mL(-1) and 27.5 s(-1), respectively. The pKa1 and pKa2 of ionizable groups of active site were 2.8 and 7.4, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: DeltaH*=33.5 kJ mol(-1), DeltaG*=70.42 kJ mol(-1) and DeltaS*=-114.37 J mol(-1) K(-1). The removal of metals resulted into complete loss of enzymatic activity and was completely recovered in the presence of 1 mM Mn2+, whereas inhibition initiated at 5 mM. The other metals like Ca2+, Zn2+ and K1+ showed no inhibition up to 7 mM, Co2+ completely inhibited the activity, while Mg2+ could not recover the initial activity up to 7 mM. So we are reporting for the first time, kinetics and thermodynamics of CMCase-Iota from G. citrina.     

Antibacterial evaluation of silver nanoparticles synthesized from lychee peel: individual versus antibiotic conjugated effects

This paper describes the extracellular synthesis of silver nanoparticles from waste part of lychee fruit (peel) and their conjugation with selected antibiotics (amoxicillin, cefixim, and streptomycin). FTIR studies revealed the reduction of metallic silver and stabilization of silver nanoparticles and their conjugates due to the presence of CO (carboxyl), OH (hydroxyl) and CH (alkanes) groups. The size of conjugated nanoparticles varied ranging from 3 to 10 nm as shown by XRD. TEM image revealed the spherical shape of biosynthesized silver nanoparticles. Conjugates of amoxicillin and cefixim showed highest antibacterial activity (147.43 and 107.95%, respectively) against Gram-negative bacteria i.e. Alcaligenes faecalis in comparison with their control counterparts. The highest reduction in MIC was noted against Gram-positive strains i.e. Enterococcus faecium (75%) and Microbacterium oxydans (75%) for amoxicillin conjugates. Anova two factor followed by two-tailed t test showed non-significant results both in case of cell leakage and protein estimation between nanoparticles and conjugates of amoxicillin, cefixime and streptomycin. In case of MDA release, non-significant difference among the test samples against the selected strains. Our study found green-synthesized silver nanoparticles as effective antibacterial bullet against both Gram positive and Gram negative bacteria, but they showed a more promising effect on conjugation with selected antibiotics against Gram negative type.     

In-vitro antibacterial, antifungal and cytotoxic activities of some coumarins and their metal complexes

A series of new antibacterial and antifungal coumarin-derived compounds and their transition metal complexes [cobalt (II), copper (II), nickel (II) and zinc (II)] have been synthesized, characterized and screened for their in vitro antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae, Bacillus cereus, Corynebacterium diphtheriae, Staphylococcus aureus and Streptococcus pyogenes bacterial strains and for in vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani, Candida glaberata. The results of these studies show the metal complexes to be more antibacterial and antifungal as compared to the uncomplexed coumarins. The brine shrimp bioassay was also carried out to study their in vitro cytotoxic properties.