Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7.

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

Loss of Ufl1/Ufbp1 in hepatocytes promotes liver pathological damage and carcinogenesis through activating mTOR signaling.

BACKGROUND: Ufm1-specific ligase 1 (Ufl1) and Ufm1-binding protein 1 (Ufbp1), as putative targets of ubiquitin-fold modifier 1 (Ufm1), have been implicated in several pathogenesis-related signaling pathways. However, little is known about their functional roles in liver disease. METHODS: Hepatocyte-specific Ufl1Δ/Δhep and Ufbp1Δ/Δhep mice were used to study their role in liver injury. Fatty liver disease and liver cancer were induced by high-fat diet (HFD) and diethylnitrosamine (DEN) administration, respectively. iTRAQ analysis was employed to screen for downstream targets affected by Ufbp1 deletion. Co-immunoprecipitation was used to determine the interactions between the Ufl1/Ufbp1 complex and the mTOR/GßL complex. RESULTS: Ufl1Δ/Δhep or Ufbp1Δ/Δhep mice exhibited hepatocyte apoptosis and mild steatosis at 2 months of age and hepatocellular ballooning, extensive fibrosis, and steatohepatitis at 6-8 months of age. More than 50% of Ufl1Δ/Δhep and Ufbp1Δ/Δhep mice developed spontaneous hepatocellular carcinoma (HCC) by 14 months of age. Moreover, Ufl1Δ/Δhep and Ufbp1Δ/Δhep mice were more susceptible to HFD-induced fatty liver and DEN-induced HCC. Mechanistically, the Ufl1/Ufbp1 complex directly interacts with the mTOR/GßL complex and attenuates mTORC1 activity. Ablation of Ufl1 or Ufbp1 in hepatocytes dissociates them from the mTOR/GßL complex and activates oncogenic mTOR signaling to drive HCC development. CONCLUSIONS: These findings reveal the potential role of Ufl1 and Ufbp1 as gatekeepers to prevent liver fibrosis and subsequent steatohepatitis and HCC development by inhibiting the mTOR pathway.

Biocontrol Capabilities of Bacillus subtilis E11 against Aspergillus flavus In Vitro and for Dried Red Chili (Capsicum annuum L.).

As a condiment with extensive nutritional value, chili is easy to be contaminated by Aspergillus flavus (A. flavus) during field, transportation, and storage. This study aimed to solve the contamination of dried red chili caused by A. flavus by inhibiting the growth of A. flavus and detoxifying aflatoxin B1 (AFB1). In this study, Bacillus subtilis E11 (B. subtilis) screened from 63 candidate antagonistic bacteria exhibited the strongest antifungal ability, which could not only inhibit 64.27% of A. flavus but could also remove 81.34% of AFB1 at 24 h. Notably, scanning electron microscopy (SEM) showed that B. subtilis E11 cells could resist a higher concentration of AFB1, and the fermentation supernatant of B. subtilis E11 could deform the mycelia of A. flavus. After 10 days of coculture with B. subtilis E11 on dried red chili inoculated with A. flavus, the mycelia of A. flavus were almost completely inhibited, and the yield of AFB1 was significantly reduced. Our study first concentrated on the use of B. subtilis as a biocontrol agent for dried red chili, which could not only enrich the resources of microbial strains for controlling A. flavus but also could provide theoretical guidance to prolong the shelf life of dried red chili.

A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila.

Ufmylation is a recently identified small ubiquitin-like modification, whose biological function and relevant cellular targets are poorly understood. Here we present evidence of a neuroprotective role for Ufmylation involving Autophagy-related gene 9 (Atg9) during Drosophila aging. The Ufm1 system ensures the health of aged neurons via Atg9 by coordinating autophagy and mTORC1, and maintaining mitochondrial homeostasis and JNK (c-Jun N-terminal kinase) activity. Neuron-specific expression of Atg9 suppresses the age-associated movement defect and lethality caused by loss of Ufmylation. Furthermore, Atg9 is identified as a conserved target of Ufm1 conjugation mediated by Ddrgk1, a critical regulator of Ufmylation. Mammalian Ddrgk1 was shown to be indispensable for the stability of endogenous Atg9A protein in mouse embryonic fibroblast (MEF) cells. Taken together, our findings might have important implications for neurodegenerative diseases in mammals.

Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target.

Immunotherapy of PD-L1/PD-1 blockage elicited impressive clinical benefits for cancer treatment. However, the relative low response and therapy resistance highlight the need to better understand the molecular regulation of PD-L1 in tumors. Here, we report that PD-L1 is a target of UFMylation. UFMylation of PD-L1 destabilizes PD-L1 by synergizing its ubiquitination. Inhibition of PD-L1 UFMylation via silencing of UFL1 or Ubiquitin-fold modifier 1 (UFM1), or the defective UFMylation of PD-L1, stabilizes the PD-L1 in multiple human and murine cancer cells, and undermines antitumor immunity in vitro and mice, respectively. Clinically, UFL1 expression was decreased in multiple cancers and lower expression of UFL1 negatively correlated with the response of anti-PD1 therapy in melanoma patients. Moreover, we identified a covalent inhibitor of UFSP2 that promoted the UFMylation activity and contributed to the combination therapy with PD-1 blockade. Our findings identified a previously unrecognized regulator of PD-L1 and highlighted UFMylation as a potential therapeutic target.

UFL1, a UFMylation E3 ligase, plays a crucial role in multiple cellular stress responses.

The UFM1 conjugation system(UFMylation)is a novel type of ubiquitin-like system that plays an indispensable role in maintaining cell homeostasis under various cellular stress. Similar to ubiquitination, UFMylation consists of a three-step enzymatic reaction with E1-like enzymes ubiquitin-like modifier activating enzyme5 (UBA5), E2-like enzymes ubiquitin-fold modifier-conjugating enzyme 1(UFC1), and E3-like ligase UFM1-specific ligase 1 (UFL1). As the only identified E3 ligase, UFL1 is responsible for specific binding and modification of the substrates to mediate numerous hormone signaling pathways and endocrine regulation under different physiological or pathological stress, such as ER stress, genotoxic stress, oncogenic stress, and inflammation. Further elucidation of the UFL1 working mechanism in multiple cellular stress responses is essential for revealing the disease pathogenesis and providing novel potential therapeutic targets. In this short review, we summarize the recent advances in novel UFL1 functions and shed light on the potential challenges ahead, thus hopefully providing a better understanding of UFMylation-mediated cellular stress.

Metabonomic analysis of human and 12 kinds of livestock mature milk.

Mature milk, as a nutrient-rich endogenous metabolite, has various beneficial effects on the human body. In order to investigate the specific nutrients provided by different dairy products to humans, we used UHPLC-Q-TOF MS to analyze the highly significantly differentially expressed metabolites in 13 species of mammalian mature milk, which were grouped into 17 major metabolite classes with 1992 metabolites based on chemical classification. KEGG shows that 5 pathways in which differentially significant metabolites are actively involved are ABC transporters, Purine metabolism, Pyrimidine metabolism, Phosphotransferase system, Galactose metabolism. The study found that pig milk and goat milk are closer to human milk and contain more nutrients that are beneficial to human health, followed by camel milk and cow milk. In the context of dairy production, the development of goat milk is more likely to meet human needs and health.

CDK5RAP3, a key defender of udder, modulates NLRP3 inflammasome activation by regulating autophagolysosome degradation in S. agalactiae-infected mastitis.

Streptococcus agalactiae, as one of the main pathogens of clinical and subclinical mastitis, affects animal welfare and leads to huge economic losses to farms due to the sharp decline in milk yield. However, both the real pathogenic mechanisms of S. agalactiae-induced mastitis and the regulator which controls the inflammation and autophagy are largely unknown. Served as a substrate of ubiquitin-like proteins of E3 ligase, CDK5RAP3 is widely involved in the regulation of multiple signaling pathways. Our findings revealed that CDK5RAP3 was significantly down-regulated in mastitis infected by S. agalactiae. Surprisingly, inflammasome activation was triggered by CDK5RAP3 knockdown: up-regulated NLRP3, IL1ß and IL6, and cleaved caspase1 promoting by NF-κB, thereby resulting in pyroptosis. Additionally, the accumulation of autophagy markers (LC3B and p62) after CDK5RAP3 knockdown suggested that the autophagolysosome degradation pathway was inhibited, thereby activating the NF-κB pathway and NLRP3 inflammasome. Hence, our findings suggest that downregulation or ablation of CDK5RAP3 inhibits autophagolysosome degradation, causes inflammation by activating the NF-κB /NLRP3 inflammasome, and triggers cell death. In conclusion, CDK5RAP3 holds the key to understanding the interaction between autophagy and immune responses, its anti-inflammatory role in this study will throw new light on the clinical drug discovery to cure S. agalactiae mastitis.

Potential of Drosophila melanogaster (fruit fly) as a dietary protein source for broilers.

This study was conducted to systematically assess and compare the fluctuations in crude protein (CP), crude fat (CF), and mineral content of staged (larva to adult) Drosophila (fruit fly) to that of a market-purchased black soldier fly larvae (BSFL) product. Results suggested that the relative CP content by dry matter ranged from 40.11% to 53.73% during Drosophila development, significantly higher (P < 0.001) than the 36.90% in BSFL. The relative CF was higher in BSFL (39.14%) compared to that of Drosophila (27.03-30.10%, P < 0.001). Although both insects contained sufficient levels of minerals to meet the dietary requirements of most animals, Drosophila overall possessed a lower content of iron, sodium, and calcium (P < 0.001) with a higher gross energy than the BSFL (P < 0.01). Comparative studies of amino acid (AA) and fatty acid (FA) profiles were further carried out among Drosophila larva (DL), pupa, and BSFL for their economic effectiveness. The AA spectra of insect larvae generally were similar except that the DL was higher in certain AA such as lysine (P < 0.01), which is an essential AA often critical for chicken growth. In contrast, the BSFL included more essential FA such as linoleic (C18:2, ω-6) and linolenic (C18:3, ω-3) acids (P < 0.01). To follow up, a husbandry trial was performed by allotting 120, 1-d-old, weight-matched, Arbor Acres broilers at random into treatment groups consisting of a low-protein diet background that contained ~20% CP supplemented with 4% BSFL and 4% or 8% DL. The average daily growth (ADG) and average daily feed intake (ADFI) of broilers, compared to the control low-protein diet, were significantly improved by feeding DL diets (P < 0.01), with better live and carcass weight and higher muscle pH (P < 0.001), which were positively correlated with the inclusion level of DL (P < 0.001). However, no differences between the control and 4% BSFL diet were observed for the performance parameters mentioned above. Moreover, all birds under our experimental setting exhibited a comparable feed conversion ratio (FCR) and were in a healthy status as indicated by the meat traits and hematological indexes within normal physiological ranges. Collectively, the findings in this study provide a theoretical basis for the further exploitation of Drosophila as potential dietary ingredients for feed production in order to meet the food challenge in the future.

Insects are regarded as one of the most promising protein sources for feed production due to its high nutritional value and low environmental cost. The objectives of this study were to analyze the dynamic nutritional composition of Drosophila (fruit fly) at various developmental phases in parallel with a commercial black soldier fly larvae (BSFL) meal, as well as to determine the effect of diets with their inclusion on broilers. Results showed that Drosophila larvae possessed a higher crude protein and a lower crude fat content when compared to the BSFL product. In the feeding trial, the performance of broilers receiving Drosophila diets was remarkably improved, with no significant influence on bird metabolic status and meat quality, except the pH of breast and thigh muscles in Drosophila diet groups being higher than that of the control group, but still in the normal range. To sum up, Drosophila meal evaluated herein has a good nutritional composition and thereby elicits a beneficial impact on the growth performance and meat production of broilers, making it a potential dietary protein source for poultry.

Life barcoded by DNA barcodes.

The modern concept of DNA-based barcoding for cataloguing biodiversity was proposed in 2003 by first adopting an approximately 600 bp fragment of the mitochondrial COI gene to compare via nucleotide alignments with known sequences from specimens previously identified by taxonomists. Other standardized regions meeting barcoding criteria then are also evolving as DNA barcodes for fast, reliable and inexpensive assessment of species composition across all forms of life, including animals, plants, fungi, bacteria and other microorganisms. Consequently, global DNA barcoding campaigns have resulted in the formation of many online workbenches and databases, such as BOLD system, as barcode references, and facilitated the development of mini-barcodes and metabarcoding strategies as important extensions of barcode techniques. Here we intend to give an overview of the characteristics and features of these barcode markers and major reference libraries existing for barcoding the planet's life, as well as to address the limitations and opportunities of DNA barcodes to an increasingly broader community of science and society.

Deficiency of Murine UFM1-Specific E3 Ligase Causes Microcephaly and Inflammation.

The UFM1 conjugation system is a Ubiquitin (Ub)-like modification system that is essential for animal development and normal physiology of multiple tissues and organs. It consists of UFM1, a Ub-like modifier, and the UFM1-specific enzymes (namely E1 enzyme UBA5, E2 enzyme UFC1 E2, and E3 ligases) that catalyze conjugation of UFM1 to its specific protein targets. Clinical studies have identified rare genetic variants in human UFM1, UBA5 and UFC1 genes that were linked to early-onset encephalopathy and defective brain development, strongly suggesting the critical role of the UFM1 system in the nervous system. Yet, the physiological function of this system in adult brain remains not defined. In this study, we investigated the role of UFM1 E3 ligase in adult mouse and found that both UFL1 and UFBP1 proteins, two components of UFM1 E3 ligase, are essential for survival of mature neurons in adult mouse. Neuron-specific deletion of either UFL1 or UFBP1 led to significant neuronal loss and elevation of inflammatory response. Interestingly, loss of one allele of UFBP1 genes caused the occurrence of seizure-like events. Our study has provided genetic evidence for the indispensable role of UFM1 E3 ligase in mature neurons and further demonstrated the importance of the UFM1 system in the nervous system.

CDK5RAP3, an essential regulator of checkpoint, interacts with RPL26 and maintains the stability of cell growth.

PURPOSE AND MATERIALS: CDK5RAP3 (CDK5 regulatory subunit associated protein 3) was originally identified as a binding protein of CDK5. It is a crucial gene controlling biological functions, such as cell proliferation, apoptosis, invasion, and metastasis. Although previous studies have also shown that CDK5RAP3 is involved in a variety of signalling pathways, however, the mechanism of CDK5RAP3 remains largely undefined. This study utilized MEFs from conditional knockout mice to inhibit CDK5RAP3 and knockdown CDK5RAP3 in MCF7 to explore the role of CDK5RAP3 in cell growth, mitosis, and cell death. RESULTS: CDK5RAP3 was found to be widely distributed throughout the centrosome, spindle, and endoplasmic reticulum, indicating that it is involved in regulating a variety of cellular activities. CDK5RAP3 deficiency resulted in instability of cell growth. CDK5RAP3 deficiency partly blocks the cell cycle in G2 /M by downregulating CDK1 (Cyclin-dependent kinase 1) and CCNB1 (Cyclin B1) expression levels. The cell proliferation rate was decreased, thereby slowing down the cell growth rate. Furthermore, the results showed that CDK5RAP3 interacts with RPL26 (ribosome protein L26) to regulate the mTOR pathway. CDK5RAP3 and RPL26 deficiency inhibited mTOR/p-mTOR protein and induce autophagy, resulting in an upregulation of the percentage of apoptosis, and the upregulated percentage of apoptosis also slowed cell growth. CONCLUSIONS: Our experiments show that CDK5RAP3 interacts with RPL26 and maintains the stability of cell growth. It shows that CDK5RAP3 plays an important role in cell growth and can be used as the target of gene medicine.

Emerging role of UFMylation in secretory cells involved in the endocrine system by maintaining ER proteostasis.

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like molecule (UBL) discovered almost two decades ago, but our knowledge about the cellular and molecular mechanisms of this novel protein post-translational modification is still very fragmentary. In this review, we first summarize the core enzymes and factors involved in the UFMylation cascade, which, similar to ubiquitin, is consecutively catalyzed by UFM1-activating enzyme 5 (UBA5), UFM1-conjugating enzyme 1 (UFC1) and UFM1-specific ligase 1 (UFL1). Inspired by the substantial implications of UFM1 machinery in the secretory pathway, we next concentrate on the puzzling role of UFMylation in maintaining ER protein homeostasis, intending to illustrate the underlying mechanisms and future perspectives. At last, given a robust ER network is a hallmark of healthy endocrine secretory cells, we emphasize the function of UFM1 modification in physiology and pathology in the context of endocrine glands pancreas and female ovaries, aiming to provide precise insight into other internal glands of the endocrine system.

A Novel TLR4-SYK Interaction Axis Plays an Essential Role in the Innate Immunity Response in Bovine Mammary Epithelial Cells.

Mammary gland epithelium, as the first line of defense for bovine mammary gland immunity, is crucial in the process of mammary glands' innate immunity, especially that of bovine mammary epithelial cells (bMECs). Our previous studies successfully marked SYK as an important candidate gene for mastitis traits via GWAS and preliminarily confirmed that SYK expression is down-regulated in bMECs with LPS (E. coli) stimulation, but its work mechanism is still unclear. In this study, for the first time, in vivo, TLR4 and SYK were colocalized and had a high correlation in mastitis mammary epithelium; protein−protein interaction results also confirmed that there was a direct interaction between them in mastitis tissue, suggesting that SYK participates in the immune regulation of the TLR4 cascade for bovine mastitis. In vitro, TLR4 also interacts with SYK in LPS (E. coli)-stimulated or GBS (S. agalactiae)-infected bMECs, respectively. Moreover, TLR4 mRNA expression and protein levels were little affected in bMECsSYK- with LPS stimulation or GBS infection, indicating that SYK is an important downstream element of the TLR4 cascade in bMECs. Interestingly, IL-1ß, IL-8, NF-κB and NLRP3 expression in LPS-stimulated or GBS-infected bMECsSYK- were significantly higher than in the control group, while AKT1 expression was down-regulated, implying that SYK could inhibit the IL-1ß, IL-8, NF-κB and NLRP3 expression and alleviate inflammation in bMECs with LPS and GBS. Taken together, our solid evidence supports that TLR4/SYK/NF-κB signal axis in bMECs regulates the innate immunity response to LPS or GBS.

Ufbp1, a Key Player of Ufm1 Conjugation System, Protects Against Ketosis-Induced Liver Injury via Suppressing Smad3 Activation.

The dairy cattle suffer from severe liver dysfunction during the pathogenesis of ketosis. The Ufm1 conjugation system is crucial for liver development and homeostasis. Ufm1 binding protein (Ufbp1) is a putative Ufm1 target and an integral component, but its role in ketosis-induced liver injury is unclear so far. The purpose of this study is to explore the key role of Ufbp1 in liver fibrosis caused by ketosis in vivo and in vitro. Liver tissues were collected from ketotic cows and Ufbp1 conditional knockout (CKO) mice in vivo. However, Ufbp1 -/- mouse embryonic fibroblast cells and Hela cells were used for in vitro validation. Subsequently, various assays were performed to reveal the underlying molecular mechanisms of the Ufbp1 protective effect. In this study, hepatic fibrosis, endoplasmic reticulum (ER) stress, and apoptosis were reported in the liver of ketotic cows, fibrotic markers (alpha-smooth muscle actin, Collagen1) and ER stress markers (glucose-regulated protein 78, CEBP homologous protein) were upregulated remarkably, and the apoptosis-related genes (Bcl2, Bax) were in line with expectations. Interestingly, Ufbp1 expression was almost disappeared, and Smad2/Smad3 protein was largely phosphorylated in the liver of ketotic cows, but Ufbp1 deletion caused Smad3 phosphorylation apparently, rather than Smad2, and elevated ER stress was observed in the CKO mice model. At the cellular level, Ufbp1 deficiency led to serious fibrotic and ER stress response, Smad3 was activated by phosphorylation significantly and then was translocated into the nucleus, whereas p-Smad2 was largely unaffected in embryonic fibroblast cells. Ufbp1 overexpression obviously suppressed Smad3 phosphorylation in Hela cells. Ufbp1 was found to be in full combination with Smad3 using endogenous immunoprecipitation. Taken together, our findings suggest that downregulation or ablation of Ufbp1 leads to Smad3 activation, elevated ER stress, and hepatocyte apoptosis, which in turn causes liver fibrosis. Ufbp1 plays a protective role in ketosis-induced liver injury.

Synergistic integration of metal nanoclusters and biomolecules as hybrid systems for therapeutic applications.

Therapeutic nanoparticles are designed to enhance efficacy, real-time monitoring, targeting accuracy, biocompatibility, biodegradability, safety, and the synergy of diagnosis and treatment of diseases by leveraging the unique physicochemical and biological properties of well-developed bio-nanomaterials. Recently, bio-inspired metal nanoclusters (NCs) consisting of several to roughly dozens of atoms (<2 nm) have attracted increasing research interest, owing to their ultrafine size, tunable fluorescent capability, good biocompatibility, variable metallic composition, and extensive surface bio-functionalization. Hybrid core-shell nanostructures that effectively incorporate unique fluorescent inorganic moieties with various biomolecules, such as proteins (enzymes, antigens, and antibodies), DNA, and specific cells, create fluorescently visualized molecular nanoparticle. The resultant nanoparticles possess combinatorial properties and synergistic efficacy, such as simplicity, active bio-responsiveness, improved applicability, and low cost, for combination therapy, such as accurate targeting, bioimaging, and enhanced therapeutic and biocatalytic effects. In contrast to larger nanoparticles, bio-inspired metal NCs allow rapid renal clearance and better pharmacokinetics in biological systems. Notably, advances in nanoscience, interfacial chemistry, and biotechnologies have further spurred researchers to explore bio-inspired metal NCs for therapeutic purposes. The current review presents a comprehensive and timely overview of various metal NCs for various therapeutic applications, with a special emphasis on the design rationale behind the use of biomolecules/cells as the main scaffolds. In the different hybrid platform, we summarize the current challenges and emerging perspectives, which are expected to offer in-depth insight into the rational design of bio-inspired metal NCs for personalized treatment and clinical translation.

DDRGK1, a crucial player of ufmylation system, is indispensable for autophagic degradation by regulating lysosomal function.

DDRGK domain-containing protein 1 (DDRGK1) is an important component of the newly discovered ufmylation system and its absence has been reported to induce extensive endoplasmic reticulum (ER) stress. Recently, emerging evidence indicates that the ufmylation system is correlated with autophagy, although the exact mechanism remains largely unknown. To explore the regulation mechanism of DDRGK1 on autophagy, in this study, we established an immortalized mouse embryonic fibroblast (MEF) cell lines harvested from the DDRGK1F/F:ROSA26-CreERT2 mice, in which DDRGK1 depletion can be induced by 4-hydroxytamoxifen (4-OHT) treatment. Here, we show that DDRGK1 deficiency in MEFs has a dual effect on autophagy, which leads to a significant accumulation of autophagosomes. On one hand, it promotes autophagy induction by impairing mTOR signaling; on the other hand, it blocks autophagy degradation by inhibiting autophagosome-lysosome fusion. This dual effect of DDRGK1 depletion on autophagy ultimately aggravates apoptosis in MEFs. Further studies reveal that DDRGK1 loss is correlated with suppressed lysosomal function, including impaired Cathepsin D (CTSD) expression, aberrant lysosomal pH, and v-ATPase accumulation, which might be a potential trigger for impairment in autophagy process. Hence, this study confirms a crucial role of DDRGK1 as an autophagy regulator by controlling lysosomal function. It may provide a theoretical basis for the treatment strategies of various physiological diseases caused by DDRGK1 deficiency.

Cdk5rap3 is essential for intestinal Paneth cell development and maintenance.

Intestinal Paneth cells are professional exocrine cells that play crucial roles in maintenance of homeostatic microbiome, modulation of mucosal immunity, and support for stem cell self-renewal. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Cdk5 activator binding protein Cdk5rap3 (also known as C53 and LZAP) was originally identified as a binding protein of Cdk5 activator p35. Although previous studies have indicated its involvement in a wide range of signaling pathways, the physiological function of Cdk5rap3 remains largely undefined. In this study, we found that Cdk5rap3 deficiency resulted in very early embryonic lethality, indicating its indispensable role in embryogenesis. To further investigate its function in the adult tissues and organs, we generated intestinal epithelial cell (IEC)-specific knockout mouse model to examine its role in intestinal development and tissue homeostasis. IEC-specific deletion of Cdk5rap3 led to nearly complete loss of Paneth cells and increased susceptibility to experimentally induced colitis. Interestingly, Cdk5rap3 deficiency resulted in downregulation of key transcription factors Gfi1 and Sox9, indicating its crucial role in Paneth cell fate specification. Furthermore, Cdk5rap3 is highly expressed in mature Paneth cells. Paneth cell-specific knockout of Cdk5rap3 caused partial loss of Paneth cells, while inducible acute deletion of Cdk5rap3 resulted in disassembly of the rough endoplasmic reticulum (RER) and abnormal zymogen granules in the mature Paneth cells, as well as loss of Paneth cells. Together, our results provide definitive evidence for the essential role of Cdk5rap3 in Paneth cell development and maintenance.

Melatonin Attenuates Peroxynitrite-Induced Meiosis Dysfunction in Porcine Oocytes.

A high level of reactive oxygen species (ROS) is widely considered one of the major causes of oocyte quality decline. Peroxynitrite is known as a powerful oxidant, which could induce multiple physical diseases. Recently, emerging pieces of evidences indicate that melatonin effectively promotes the development of oocytes, although the specific work mechanism remains to be further clarified. In this study, it was shown that peroxynitrite increased the level of ROS in porcine oocytes, which induced the apoptosis of oocytes, thereby leading to the obstruction of spindle assembly, depolymerization of actin, and decrease of polar body expulsion. These negative effects contributed to the failure of meiosis and ultimately blocked the maturation of porcine oocytes. As expected, it was found that melatonin effectively removed the accumulated ROS in oocytes, preventing oocytes from peroxynitrite-induced oocyte maturation failure, which might provide a novel approach to improve female livestock reproduction and cure female infertility in clinical practice.

CDK5RAP3, a Novel Nucleoplasmic Shuttle, Deeply Regulates HSF1-Mediated Heat Stress Response and Protects Mammary Epithelial Cells from Heat Injury.

CDK5RAP3 was regarded as the most significant regulator of cellular responses against heat stress, which is associated with dysfunctions of the immune system and animal susceptibility to disease. Despite this, little known about how CDK5RAP3 regulates heat stress response. In this study, CDK5RAP3 conditional Knockout (CKO) mice, CDK5RAP3-/- mouse embryo fibroblasts (MEFs) and bovine mammary epithelial cells (BMECs) were used as an in vitro and in vivo model, respectively to reveal the role of CDK5RAP3 in regulating the heat stress response. The deletion of CDK5RAP3 unexpectedly caused animal lethality after 1.5-h heat stimulations. Furthermore, BMECs were re-cultured for eight hours after heat stress and was found that the expression of CDK5RAP3 and HSPs showed a similar fluctuating pattern of increase (0-2, 4-6 h) and decrease (2-4, 6-8 h). In addition to the remarkably enhanced expression of heat shock protein, apoptosis rate and endoplasmic reticulum stress, the deletion of CDK5RAP3 also affected nucleoplasmic translocation and trimer formation of heat shock factor 1 (HSF1). These programs were further confirmed in the mammary gland of CDK5RAP3 CKO mice and CDK5RAP3-/- MEFs as well. Interestingly, genetic silencing of HSF1 downregulated CDK5RAP3 expression in BMECs. Immunostaining and immunoprecipitation studies suggested a physical interaction between CDK5RAP3 and HSF1 being co-localized in the cytoplasm and nucleus. Besides, CDK5RAP3 also interacted with HSP90, suggesting an operative machinery at both transcriptional level and protein functionality of HSP90 per se. Together, our findings suggested that CDK5RAP3 works like a novel nucleoplasmic shuttle or molecular chaperone, deeply participating in HSF1-mediated heat stress response and protecting cells from heat injury.