Dormancy is a physiological state that confers winter hardiness to and orchestrates phenological phase progression in temperate perennial plants. Weather fluctuations caused by climate change increasingly disturb dormancy onset and release in many plant species including tree crops leading to aberrant growth, flowering, and fruiting. Currently, research in this field is impeded by the lack of affordable non-invasive methods for on-line monitoring of dormancy. We report on an automatic framework for low-cost, long-term, and scalable dormancy studies in deciduous plants. The proposed method is based on continuous near-field sensing of the photosynthetic activity of shoots via pulse-amplitude modulated chlorophyll fluorescence sensors connected remotely to a data processing system. The resulting high-resolution time series of JIP-test parameters indicative of the responsiveness of the photosynthetic apparatus to environmental stimuli are subjected to frequency-domain analysis. The proposed approach allows to overcome the variance coming from diurnal changes of insolation and to derive estimations on the depth of dormancy. Our approach was validated over three seasons in an experimental apple (Malus × domestica Borkh.) orchard by collating the non-invasive estimations with the results of traditional methods (growing of the cuttings obtained from the tress at different phases of dormancy) and the output of commonly used chilling requirement models. We discuss the advantages of the proposed monitoring framework such as prompt detection of freeze damages along with its potential limitations.

Abstract: Passive acoustic monitoring (PAM) is a non-invasive technique to supervise the wildlife. Acoustic surveillance is preferable in some situation such as in the case of marine mammals, when the animals spend most of their time underwater, making it hard to obtain their images. Machine learning is very useful for PAM, for example, to identify species based on audio recordings. But some care should be taken to evaluate the capability of a system. We define PAM-filters as the creation of the experimental protocols according to the dates and locations of the recordings, aiming to avoid the use of the same individuals, noise and recording devices in both training and test sets. A random division of a database present accuracies much higher than accuracies obtained with protocols generated with PAM-filter. Although we use the animal vocalizations, in our method we convert the audio into spectrogram images, after that, we describe the images using the texture. Those are well-known techniques for audio classification, and they have already been used for species classification. Also, we perform statistical tests to demonstrate the significant difference between accuracies generated with and without PAM-filters with several well-known classifiers. The configuration of our experimental protocols and the database were made available online.

Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology and medicine. The current study focuses on generating a construct to express a compact Cas9 nuclease (AnoCas9) from the thermophilic microorganism Anoxybacillus flavithermus. Next, distinctive AnoCas9 properties are investigated. AnoCas9 gene is expressed in E.coli producing a polypeptide fused with the maltose-binding protein (MBP-AnoCas9). His-Tag in its structure enables purification using metal-chelate chromatography followed by the cleavage of the polypeptide by TEV protease. Bioinformatical analysis of the CRISPR array found in the genome of an Anoxybacillus flavithermus strain helps predict a functional PAM sequence for AnoCas9, which is supported by in vitro experiments. The purified protein demonstrates nuclease activity in the presence of crRNA:tracrRNA duplex in the 37-60 °С range, with maximum activity observed at 45-55 °С. The analysis of FAM-labeled dsDNA substrate cleavage has allowed us to determine the functional AnoCas9 PAM motif as 5’-NNNNCDAA-3’. Thus, AnoCas9 adds to the repertoire of thermophilic Cas9 effectors and its properties suggest application in areas requiring the presence of thermostable CRISPR/Cas systems.

We have investigated the individual and combined effects of temperature and light on the physi-ology of the diatom Phaeodactylum tricornutum, a model organism widely used for studies on diatom physiology and ecology and biofuel production. Our results show that the combination of changes in temperature and light intensity affects growth rates, the content of pigments and active photosystems, photosynthetic efficiency, lipid production and fatty acid composition of P. tricornutum. Measurements of the maximum electron transport rate (rETRmax) and rETR at maximum PAR, confirmed that in P. tricornutum light sensitivity is substantially higher as the growth temperature increases under light/dark cycles of two light intensities (25-60 µmol m-2 s-1) but it is just the opposite under continuous light (25 µmol m-2 s-1). Furthermore, higher rETRmax were observed at higher ir-radiance (either in intensity or in a continuous light time regime) at the two temperatures tested. On the other hand, increasing light intensity amplifies the observed effect of temperature on photo-system I (PSI) activity in light/dark regimes, but not in continuous light conditions, leading to a greater deficiency in PSI activity, due to limitations in the electron supply to this photosystem. Moreover, the change in the culture temperature from 20°C to 25°C triggers an increase in the number and size of cytoplasmic lipid droplets under conditions of increased light intensity, but even more drastically under continuous illumination. It should be noted that the combination of a temperature of 25°C and continuous illumination in P. tricornutum cell cultures causes a drastic increase of triacylglycerides, and a change in the composition of total fatty acids that is optimal for a possible use as biodiesel.

Photoacoustic technology is a promising tool to provide morphological and functional information in biomedical research. To enhance the imaging efficiency, the reported photoacoustic probes have been designed coaxially involving complicated optical/acoustic prisms to bypass the opaque piezoelectric layer of ultrasound transducers, but leading to bulky probes and hindering the applications in limited space. Though the emergence of transparent piezoelectric materials helps to save the effort on the coaxial design, the reported transparent ultrasound transducers were still bulky. In this work, a miniature photoacoustic probe with an outer diameter of 4 mm was developed, in which an acoustic stack was made of the combination of transparent piezoelectric material and gradient-index lens as a backing layer. The transparent ultrasound transducer exhibited a high center frequency of ~47 MHz and a -6 dB bandwidth of 29.4%, which could be easily assembled with a pigtailed ferrule of a single-mode fiber. The multi-functional capability of the probe was successfully validated through the experiments of fluid flow sensing and photoacoustic imaging.

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

Keywords: Requirements prioritization, Next release plan, Software product planning, Decision support, MoSCoW, AHP, K-Menas, GMM, BIRCH, PAM, Hierarchical, Clustering, Clusters Evaluation