Amino acid metabolism and MAP kinase signaling pathway play opposite roles in the regulation of ethanol production during fermentation of sugarcane molasses in budding yeast.

Sugarcane molasses is one of the main raw materials for bioethanol production, and Saccharomyces cerevisiae is the major biofuel-producing organism. In this study, a batch fermentation model has been used to examine ethanol titers of deletion mutants for all yeast nonessential genes in this yeast genome. A total of 42 genes are identified to be involved in ethanol production during fermentation of sugarcane molasses. Deletion mutants of seventeen genes show increased ethanol titers, while deletion mutants for twenty-five genes exhibit reduced ethanol titers. Two MAP kinases Hog1 and Kss1 controlling the high osmolarity and glycerol (HOG) signaling and the filamentous growth, respectively, are negatively involved in the regulation of ethanol production. In addition, twelve genes involved in amino acid metabolism are crucial for ethanol production during fermentation. Our findings provide novel targets and strategies for genetically engineering industrial yeast strains to improve ethanol titer during fermentation of sugarcane molasses.

Identification of the Beta Subunit Fas1p of Fatty Acid Synthetase as an Interacting Partner of Yeast Calcium/Calmodulin-Dependent Protein Kinase Cmk2p Through Mass Spectrometry Analysis.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is a mediator of calcium signals and regulates fatty acid metabolism in mammalian cells. Cmk2p is a yeast homolog of CaMKII and functions as a negative regulator of calcium signaling. However, its substrates remain to be identified. Combination of immunoprecipitation (IP) and mass spectrometry has been proven to be very useful for identification of interacting partner proteins and interactome. In this study, through these approaches, we have identified 65 and 110 potential Cmk2p-interacting proteins in yeast cells in the absence or presence of calcium stress, respectively. In yeast cells expressing both CMK2-HA and FAS1-GFP fusion proteins, in the absence or presence of calcium stress, less amounts of FAS1-GFP proteins are present in cell lysates after IP with anti-HA antibody than cell lysates before IP, while FAS1-GFP proteins are detected on both types of IP beads. However, as an internal control, similar amounts of Pgk1p proteins were detected in both after-IP and before-IP cell lysates but not on the IP beads. Therefore, our biochemical analysis demonstrates that the ß subunit Fas1p of fatty acid synthetase interacts with Cmk2p in yeast cells independent of calcium stress. It is also interesting to note that, in addition to the expected 52-kDa CMK2-HA band, a faster-moving 48-kDa CMK2-HA band is present in the calcium-stressed cell lysate but not in the cell lysate without calcium stress. Our data would provide important clues for understanding the functions of CaMKII in the regulation of fatty acid metabolism as well as related diseases such as cancers, diabetes, and obesity.

A glycosylated Phr1 protein is induced by calcium stress and its expression is positively controlled by the calcium/calcineurin signaling transcription factor Crz1 in Candida albicans.

As one of the most important human fungal pathogens, Candida albicans senses and adapts to host niches with different pH values through the pH-responsive Rim101 pathway. Its transcription factor Rim101 activates the expression of alkaline pH-induced genes including PHR1 that encodes a glycosylphosphatidylinsitol-anchored ß(1,3)-glucanosyltransferase critical for hyphal wall formation. The calcium/calcineurin signaling pathway is mediated by the transcription factor Crz1 in yeasts and other lower eukaryotes. Here we report that deletion of PHR1 leads to calcium sensitivity of C. albicans cells. In addition, expression of Phr1 is induced by calcium stress and under the control of Crz1 in C. albicans. EMSA assay demonstrates that Crz1 binds to one CDRE element in the PHR1 promoter. Alkaline treatment induces two species of glycosylated Phr1 proteins with different degrees of glycosylation, which is independent of Crz1. In contrast, only one species of Phr1 protein with a low degree of glycosylation is induced by calcium stress in a Crz1-dependent fashion. Therefore, we have provided an evidence that regulation of cell wall remodeling is integrated through differential degrees of Phr1 glycosylation by both the pH-regulated Rim101 pathway and the calcium/calcineurin signaling pathway in C. albicans. Video Abstract.

Generating Functional Multicellular Organoids from Human Placenta Villi.

The interaction between trophoblasts, stroma cells, and immune cells at the maternal-fetal interface constitutes the functional units of the placenta, which is crucial for successful pregnancy outcomes. However, the investigation of this intricate interplay is restricted due to the absence of efficient experimental models. To address this challenge, a robust, reliable methodology for generating placenta villi organoids (PVOs) from early, late, or diseased pregnancies using air-liquid surface culture is developed. PVOs contain cytotrophoblasts that can self-renew and differentiate directly, along with stromal elements that retain native immune cells. Analysis of scRNA sequencing and WES data reveals that PVOs faithfully recapitulate the cellular components and genetic alterations of the corresponding source tissue. Additionally, PVOs derived from patients with preeclampsia exhibit specific pathological features such as inflammation, antiangiogenic imbalance, and decreased syncytin expression. The PVO-based propagation of primary placenta villi should enable a deeper investigation of placenta development and exploration of the underlying pathogenesis and therapeutics of placenta-originated diseases.

A novel strategy of engineering genetically encoded probe for ultrasensitive sensing Hg2+ with unusual planar trigonometric coordination configuration.

At present, few genetically encoded fluorescent probes are currently available for the analysis of toxic heavy metal ions, and most have poor performance that cannot meet the requirements of sensitive and dynamic detection in living cells. In this study, we designed a single fluorescent protein-based probe sfGFP-MerBD, which can specifically response to Hg2+ with high binding affinity and wide dynamic range. More importantly, the developing probe can timely and reversibly monitor changes of Hg2+ concentration in living mammalian cells. The excellent performance of this probe is largely due to the recognition element of the probe, MerBD, which adopts an unusual planar trigonometric coordination configuration with Hg2+, and the coordination can cause enough conformational change to influence the fluorescence of skeleton protein sfGFP coupled with it. The small peptide MerBD was delicately designed based on the three-dimensional structure of metalloprotein MerR. This novel design strategy solves the challenging problems that there are few natural functional proteins in the process of constructing fluorescent probes for toxic metal ions and some functional proteins cannot be directly used as recognition elements. Based on the new strategy, more genetically encoded fluorescent probes of toxic heavy metal ions can be efficiently constructed and applied in the future.

Rapid field testing of mercury pollution by designed fluorescent biosensor and its cells-alginate hydrogel-based paper assay.

With increasing industrial activities, mercury has been largely discharged into environment and caused serious environmental problems. The growing level of mercury pollution has become a huge threat to human health due to its significant biotoxicity. Therefore, the simple and fast means for on-site monitoring discharged mercury pollution are highly necessary to protect human beings from its pernicious effects in time. Herein, a "turn off" fluorescent biosensor (mCherry L199C) for sensing Hg2+ was successfully designed based on direct modification of the chromophore environment of fluorescent protein mCherry. For rapid screening and characterization, the designed variant of mCherry (mCherry L199C) was directly expressed on outer-membrane of  Escherichia coli cells by cell surface display technique. The fluorescent biosensor was characterized to have favorable response to Hg2+ at micromole level among other metal ions and over a broad pH range. Further, the cells of the fluorescent biosensor were encapsulated in alginate hydrogel to develop the cells-alginate hydrogel-based paper. The cells-alginate hydrogel-based paper could detect mercury pollution in 5 min with simple operation process and inexpensive equipment, and it could keep fluorescence and activity stable at 4 °C for 24 hr, which would be a high-throughput screening tool in preliminarily reporting the presence of mercury pollution in natural setting.

A signal-amplified whole-cell biosensor for sensitive detection of Hg2+ based on Hg2+-enhanced reporter module.

A signal-amplified mercury sensing biosensor with desired sensitivity was developed through firstly using the GFP mutant with fluorescence increasing response towards Hg2+ as the reporter module. The developed biosensor showed response for Hg2+ in a relatively wide range of 1-10,000 nmol/L, and the detection limit was improved one or two orders of magnitude in comparison with most metal-sensing biosensors in similar constructs. In addition, the biosensor could distinguish Hg2+ easily from multiple metal ions and displayed strong adaptability to extensive pH conditions (pH 4.0-10.0). More importantly, the developed biosensor was able to provide an initial assessment of Hg2+ spiked in the environmental water with the recoveries between 85.70% and 112.50%. The signal-amplified strategy performed by the modified reporter module will be widely applicable to many other whole-cell biosensors, meeting the practical requirements with sufficient sensing performance.

[Phacoemulsification cataract surgery with different cumulative energy composite parameters in patients with type 2 diabetes mellitus:therapeutic effect and complications].

OBJECTIVE: To evaluate the effect of different cumulative energy composite parameters on the outcomes of phacoemulsification cataract surgery in patients with type 2 diabetes mellitus. METHODS: A total of 252 patients with cataract (involving 252 eyes) and type 2 diabetes mellitus received phacoemulsification cataract surgery in our hospital between January, 2017 and June, 2019. The patients were divided into group A (150 cases) and group B (102 cases) for cataract phacoemulsification with cumulative energy composite parameters of 8 and 10, respectively, and 90 nondiabetic patients received cataract phacoemulsification with a cumulative energy composite parameters of 10 served as the control. The macular thickness, best corrected visual acuity, visual acuity, and postoperative leakage in the 3 groups were evaluated at 1 week, 1 month, and 3 months after the surgery. RESULTS: The visual acuity was significantly improved after phacoemulsification better in all the 3 groups. At 3 months after the surgery, the proportions of patients with visual acuity ratio < 0.1 or >1.0, macular thickness, best corrected visual acuity and permeability differed significantly between groups A and B (P < 0.05), but not between group A and the control group (P > 0.05). At 1 month and 3 months after the surgery, the proportion of patients with visual acuity ratio < 0.1 was significantly lower and the rate of visual acuity ratio >1.0 was higher in group A than in group B. At 1 month after the operation, the total leakage rate in group A (31.1%) was higher than that in the control group (21.1%) but comparable with that in group B; at 3 months, the total leakage rates were significantly lower in group A than in group B (10.0% vs 32.4%, P < 0.05), and the leakage resulted mainly from local and diffuse permeation. CONCLUSIONS: Phacoemulsification can effectively improve the visual acuity of cataract patients especially in non-diabetic patients. A lower cumulative energy composite parameter achieves better outcomes in type 2 diabetic patients with cataract. The macular thickness, local infiltration and diffuse leakage can be used as indicators for assessing visual recovery and stabilization after phacoemulsification.