Multiple Leptin Signalling Pathways in the Control of Metabolism and Fertility: A Means to Different Ends?

The adipocyte-derived ‘satiety promoting’ hormone, leptin, has been identified as a key central regulator of body weight and fertility, such that its absence leads to obesity and infertility. Plasma leptin levels reflect body adiposity, and therefore act as an ‘adipostat’, whereby low leptin levels reflect a state of low body adiposity (under-nutrition/starvation) and elevated leptin levels reflect a state of high body adiposity (over-nutrition/obesity). While genetic leptin deficiency is rare, obesity-related leptin resistance is becoming increasingly common. In the absence of adequate leptin sensitivity, leptin is unable to exert its ‘anti-obesity’ effects, thereby exacerbating obesity. Furthermore, extreme leptin resistance and consequent low or absent leptin signalling resembles a state of starvation and can thus lead to infertility. However, leptin resistance occurs on a spectrum, and it is possible to be resistant to leptin’s metabolic effects while retaining leptin’s permissive effects on fertility. This may be because leptin exerts its modulatory effects on energy homeostasis and reproductive function through discrete intracellular signalling pathways, and these pathways are differentially affected by the molecules that promote leptin resistance. This review discusses the potential mechanisms that enable leptin to exert differential control over metabolic and reproductive function in the contexts of healthy leptin signalling and of diet-induced leptin resistance.     

Dadaist2: A Toolkit to Automate and Simplify Statistical Analysis and Plotting of Metabarcoding Experiments

The taxonomic composition of microbial communities can be assessed using universal marker amplicon sequencing. The most common taxonomic markers are the 16S rDNA for bacterial communities and the internal transcribed spacer (ITS) region for fungal communities, but various other markers are used for barcoding eukaryotes. A crucial step in the bioinformatic analysis of amplicon sequences is the identification of representative sequences. This can be achieved using a clustering approach or by denoising raw sequencing reads. DADA2 is a widely adopted algorithm, released as an R library, that denoises marker-specific amplicons from next-generation sequencing and produces a set of representative sequences referred to as ‘Amplicon Sequence Variants’ (ASV). Here, we present Dadaist2, a modular pipeline, providing a complete suite for the analysis that ranges from raw sequencing reads to the statistics of numerical ecology. Dadaist2 implements a new approach that is specifically optimised for amplicons with variable lengths, such as the fungal ITS. The pipeline focuses on streamlining the data flow from the command line to R, with multiple options for statistical analysis and plotting, both interactive and automatic.     

Non-Invasive Preimplantation Genetic Testing for Aneuploidy and the Mystery of Genetic Material: A Review Article

This review focuses on recent findings in the preimplantation genetic testing (PGT) of embryos. Different preimplantation genetic tests are presented along with different genetic materials and their analysis. Original material concerning preimplantation genetic testing for aneuploidy (PGT-A) was sourced by searching the PubMed and ScienceDirect databases in October and November 2021. The searches comprised keywords such as ‘preimplantation’, ‘cfDNA’; ‘miRNA’, ‘PGT-A’, ‘niPGT-A’, ‘aneuploidy’, ‘mosaicism’, ‘blastocyst biopsy’, ‘blastocentesis’, ‘blastocoel fluid’, ‘NGS’, ‘FISH’, and ‘aCGH’. Non-invasive PGT-A (niPGT-A) is a novel approach to the genetic analysis of embryos. The premise is that the genetic material in the spent embryo culture media (SECM) corresponds to the genetic material in the embryo cells. The limitations of niPGT-A are a lower quantity and lesser quality of the cell-free genetic material, and its unknown origin. The concordance rate varies when compared to invasive PGT-A. Some authors have also hypothesized that mosaicism and aneuploid cells are preferentially excluded from the embryo during early development. Cell-free genetic material is readily available in the spent embryo culture media, which provides an easier, more economic, and safer extraction of genetic material for analysis. The sampling of the SECM and DNA extraction and amplification must be optimized. The origin of the cell-free media, the percentage of apoptotic events, and the levels of DNA contamination are currently unknown; these topics need to be further investigated.     

Comparison of two data interpretation programs in automated blood cell counters

Two data interpretation programs incorporated into two different automated blood cell counters were evaluated. The programs analysed size distribution histograms for screening for abnormal specimens. The messages given by the programs were ‘normal’, ‘abnormal’ and ‘suspect’. ‘Abnormal’ messages meant abnormalities in the values of the measured parameters. ‘Suspect’ messages were given when the histograms showed either a possibility of the existence of abnormal blood cells, or an abnormal level of such cells; re-examination by a manual method was necessary for such specimens. For reference, the manual differential was done. The false-negative rate for each kind of message was less than 10% and efficiency was about 90%. These programs were helpful in avoiding unnecessary manual observations in reducing the workload in a routine clinical laboratory.     

Wnt Modulation Enhances Otic Differentiation by Facilitating the Enucleation Process but Develops Unnecessary Cardiac Structures

Otic organoids have the potential to resolve current challenges in hearing loss research. The reproduction of the delicate and complex structure of the mammalian cochlea using organoids requires high efficiency and specificity. Recent attempts to strengthen otic organoids have focused on the effects of the Wnt signaling pathway on stem cell differentiation. One important aspect of this is the evaluation of undesirable effects of differentiation after Wnt activation. In the present study, we differentiated mouse embryonic stem cell embryoid bodies (EB) into otic organoids and observed two morphologies with different cell fates. EBs that underwent a core ejection process, or ‘enucleation,’ were similar to previously reported inner ear organoids. Meanwhile, EBs that retained their core demonstrated features characteristic of neural organoids. The application of a Wnt agonist during the maturation phase increased enucleation, as well as otic organoid formation, in turn leading to sensory hair cell-like cell generation. However, with a longer incubation period, Wnt activation also led to EBs with ‘beating’ organoids that exhibited spontaneous movement. This observation emphasizes the necessity of optimizing Wnt enhancement for the differentiation of specific cells, such as those found in the inner ear.     

Physiological and Molecular Analysis Reveals the Differences of Photosynthesis between Colored and Green Leaf Poplars

Leaf coloration changes evoke different photosynthetic responses among different poplar cultivars. The aim of this study is to investigate the photosynthetic difference between a red leaf cultivar (ZHP) and a green leaf (L2025) cultivar of Populus deltoides. In this study, ‘ZHP’ exhibited wide ranges and huge potential for absorption and utilization of light energy and CO₂ concentration which were similar to those in ‘L2025’ and even showed a stronger absorption for weak light. However, with the increasing light intensity and CO₂ concentration, the photosynthetic capacity in both ‘L2025’ and ‘ZHP’ was gradually restricted, and the net photosynthetic rate (Pn) in ‘ZHP’ was significantly lower than that in ‘L2025’under high light or high CO₂ conditions, which was mainly attributed to stomatal regulation and different photosynthetic efficiency (including the light energy utilization efficiency and photosynthetic CO₂ assimilation efficiency) in these two poplars. Moreover, the higher anthocyanin content in ‘ZHP’ than that in ‘L2025’ was considered to be closely related to the decreased photosynthetic efficiency in ‘ZHP’. According to the results from the JIP-test, the capture efficiency of the reaction center for light energy in ‘L2025’ was significantly higher than that in ‘ZHP’. Interestingly, the higher levels of light quantum caused relatively higher accumulation of Q_A^- in ‘L2025’, which blocked the electron transport and weakened the photosystem II (PSII) performance as compared with ‘ZHP’; however, the decreased capture of light quantum also could not promote the utilization of light energy, which was the key to the low photosynthetic efficiency in ‘ZHP’. The differential expressions of a series of photosynthesis-related genes further promoted these specific photosynthetic processes between ‘L2025’ and ‘ZHP’.     

PSII Activity Was Inhibited at Flowering Stage with Developing Black Bracts of Oat

The color of bracts generally turns yellow or black from green during cereal grain development. However, the impact of these phenotypic changes on photosynthetic physiology during black bract formation remains unclear. Two oat cultivars (Avena sativa L.), ‘Triple Crown’ and ‘Qinghai 444’, with yellow and black bracts, respectively, were found to both have green bracts at the heading stage, but started to turn black at the flowering stage and become blackened at the milk stage for ‘Qinghai 444’. Their photosynthetic characteristics were analyzed and compared, and the key genes, proteins and regulatory pathways affecting photosynthetic physiology were determined in ‘Triple Crown’ and ‘Qinghai 444’ bracts. The results show that the actual PSII photochemical efficiency and PSII electron transfer rate of ‘Qinghai 444’ bracts had no significant changes at the heading and milk stages but decreased significantly (p < 0.05) at the flowering stage compared with ‘Triple Crown’. The chlorophyll content decreased, the LHCII involved in the assembly of supercomplexes in the thylakoid membrane was inhibited, and the expression of Lhcb1 and Lhcb5 was downregulated at the flowering stage. During this critical stage, the expression of Bh4 and C4H was upregulated, and the biosynthetic pathway of p-coumaric acid using tyrosine and phenylalanine as precursors was also enhanced. Moreover, the key upregulated genes (CHS, CHI and F3H) of anthocyanin biosynthesis might complement the impaired PSII activity until recovered at the milk stage. These findings provide a new insight into how photosynthesis alters during the process of oat bract color transition to black.     

Comparative Proteomic Analysis of Mature Pollen in Triploid and Diploid Populus deltoides

Ploidy affects plant growth vigor and cell size, but the relative effects of pollen fertility and allergenicity between triploid and diploid have not been systematically examined. Here we performed comparative analyses of fertility, proteome, and abundances of putative allergenic proteins of pollen in triploid poplar ‘ZhongHuai1’ (‘ZH1’, triploid) and ‘ZhongHuai2’ (‘ZH2’, diploid) generated from the same parents. The mature pollen was sterile in triploid poplar ‘ZH1’. By applying two-dimensional gel electrophoresis (2-DE), a total of 72 differentially expressed protein spots (DEPs) were detected in triploid poplar pollen. Among them, 24 upregulated and 43 downregulated proteins were identified in triploid poplar pollen using matrix-assisted laser desorption/ionisation coupled with time of-flight tandem mass spectrometer analysis (MALDI-TOF/TOF MS/MS). The main functions of these DEPs were related with “S-adenosylmethionine metabolism”, “actin cytoskeleton organization”, or “translational elongation”. The infertility of triploid poplar pollen might be related to its abnormal cytoskeletal system. In addition, the abundances of previously identified 28 putative allergenic proteins were compared among three poplar varieties (‘ZH1’, ‘ZH2’, and ‘2KEN8‘). Most putative allergenic proteins were downregulated in triploid poplar pollen. This work provides an insight into understanding the protein regulation mechanism of pollen infertility and low allergenicity in triploid poplar, and gives a clue to improving poplar polyploidy breeding and decreasing the pollen allergenicity.     

Chilling Requirement Validation and Physiological and Molecular Responses of the Bud Endodormancy Release in Paeonia lactiflora ‘Meiju’

The introduction of herbaceous peony (Paeonia lactiflora Pall.) in low-latitude areas is of great significance to expand the landscape application of this world-famous ornamental. With the hazards of climate warming, warm winters occurs frequently, which makes many excellent northern herbaceous peony cultivars unable to meet their chilling requirements (CR) and leads to their poor growth and flowering in southern China. Exploring the endodormancy release mechanism of underground buds is crucial for improving low-CR cultivar screening and breeding. A systematic study was conducted on P. lactiflora ‘Meiju’, a screened cultivar with a typical low-CR trait introduced from northern China, at the morphological, physiological and molecular levels. The CR value of ‘Meiju’ was further verified as 677.5 CUs based on the UT model and morphological observation. As a kind of signal transducer, reactive oxygen species (ROS) released a signal to enter dormancy, which led to corresponding changes in carbohydrate and hormone metabolism in buds, thus promoting underground buds to acquire strong cold resistance and enter endodormancy. The expression of important genes related to ABA metabolism, such as NCED3, PP2C, CBF4 and ABF2, reached peaks at the critical stage of endodormancy release (9 January) and then decreased rapidly; the expression of the GA2ox8 gene related to GA synthesis increased significantly in the early stage of endodormancy release and decreased rapidly after the release of ecodormancy (23 January). Cytological observation showed that the period when the sugar and starch contents decreased and the ABA/GA ratio decreased was when ‘Meiju’ bud endodormancy was released. This study reveals the endodormancy regulation mechanism of ‘Meiju’ buds with the low-CR trait, which lays a theoretical foundation for breeding new herbaceous peony cultivars with the low-CR trait.     

Phosphate Metabolism and Pathophysiology in Parathyroid Disorders and Endocrine Tumors

The advent of new insights into phosphate metabolism must urge the endocrinologist to rethink the pathophysiology of widespread disorders, such as primary hyperparathyroidism, and also of rarer endocrine metabolic bone diseases, such as hypoparathyroidism and tumor-induced hypophosphatemia. These rare diseases of mineral metabolism have been and will be a precious source of new information about phosphate and other minerals in the coming years. The parathyroid glands, the kidneys, and the intestine are the main organs affecting phosphate levels in the blood and urine. Parathyroid disorders, renal tubule defects, or phosphatonin-producing tumors might be unveiled from alterations of such a simple and inexpensive mineral as serum phosphate. This review will present all these disorders from a ‘phosphate perspective’.     

Electrostatic complexation of polyelectrolyte and magnetic nanoparticles: from wild clustering to controllable magnetic wires

We present the electrostatic complexation between polyelectrolytes and charged nanoparticles. The nanoparticles in solution are γ-Fe₂O₃ (maghemite) spheres with 8.3 nm diameter and anionic surface charges. The complexation was monitored using three different formulation pathways such as direct mixing, dilution, and dialysis. In the first process, the hybrids were obtained by mixing stock solutions of polymers and nanoparticles. A ‘destabilization state’ with sharp and intense maximum aggregation was found at charges stoichiometry (isoelectric point). While on the two sides of the isoelectric point, ‘long-lived stable clusters state’ (arrested states) were observed. Dilution and dialysis processes were based on controlled desalting kinetics according to methods developed in molecular biology. Under an external magnetic field (B = 0.3 T), from dialysis at isoelectric point and at arrested states, cationic polyelectrolytes can ‘paste’ these magnetic nanoparticles (NPs) together to yield irregular aggregates (size of 100 μm) and regular rod-like aggregates, respectively. These straight magnetic wires were fabricated with diameters around 200 nm and lengths comprised between 1 μm and 0.5 mm. The wires can have either positive or negative charges on their surface. After analyzing their orientational behavior under an external rotating field, we also showed that the wires made from different polyelectrolytes have the same magnetic property. The recipe used a wide range of polyelectrolytes thereby enhancing the versatility and applied potentialities of the method. This simple and general approach presents significant perspective for the fabrication of hybrid functional materials.     

Positioning laboratory automation for today's dynamic climate

Laboratory automation has existed and matured at Eli Lilly and Company for well over a decade. The author''s section serves as a developer of laboratory automation systems for customers within Lilly and embodies ‘robotic friendly’ laboratories with highly technical and experienced personnel. With several systems showing signs of age, second generation ‘smart systems’ have been developed and delivered during the last three years. These systems were built with an ideology different from previous systems. Upon their delivery, the ‘smart systems’ met the customer''s functional requirements but the overall acceptance of this ideology is still being debated due to the perception of failure. Much of this perception can be attributed to the delivery of a system heavily dependent on system maintenance, something totally unexpected by the customer. This paper discusses the ideology of a‘smart systems’ and the results following implementation. The events that led to the review and subsequent departure of the ‘smart systems’ ideology are also described.