Spatial memory requires hypocretins to elevate medial entorhinal gamma oscillations.

The hypocretin (Hcrt) (also known as orexin) neuropeptidic wakefulness-promoting system is implicated in the regulation of spatial memory, but its specific role and mechanisms remain poorly understood. In this study, we revealed the innervation of the medial entorhinal cortex (MEC) by Hcrt neurons in mice. Using the genetically encoded G-protein-coupled receptor activation-based Hcrt sensor, we observed a significant increase in Hcrt levels in the MEC during novel object-place exploration. We identified the function of Hcrt at presynaptic glutamatergic terminals, where it recruits fast-spiking parvalbumin-positive neurons and promotes gamma oscillations. Bidirectional manipulations of Hcrt neurons' projections from the lateral hypothalamus (LHHcrt) to MEC revealed the essential role of this pathway in regulating object-place memory encoding, but not recall, through the modulation of gamma oscillations. Our findings highlight the significance of the LHHcrt-MEC circuitry in supporting spatial memory and reveal a unique neural basis for the hypothalamic regulation of spatial memory.

Perillaldehyde: A promising antibacterial agent for the treatment of pneumonia caused by Acinetobacter baumannii infection.

Perillaldehyde is a monoterpene compound mainly from the medicinal plant Perilla frutescens (L.) Britt., which has hypolipidemic, antioxidant, antibacterial and anti-inflammatory functions. In this investigation, we discovered that Perillaldehyde had powerful antimicrobial activity against Acinetobacter baumannii 5F1, and its minimum inhibitory concentration was 287.08 µg/mL. A. baumannii is a conditionally pathogenic bacterium with a high clinical resistance rate and is a major source of hospital infections, especially in intensive care units, which is one of the main causes of pneumonia. Inflammatory immune response is characteristic of pneumonia caused by A. baumannii infection. The results of our in vitro experiments indicate that Perillaldehyde disrupts the cell membrane of A. baumannii 5F1 and inhibits its quorum sensing to inhibit biofilm formation, among other effects. With an experimental model of murine pneumonia, we investigated that Perillaldehyde decreased NLRP3 inflammasome activation and TNF-α expression in lung tissues by inhibiting the NF-κB pathway, and also impacted MAPKs protein signaling pathway through the activation of TLR4. Notably, the use of high doses of Perillaldehyde for the treatment of pneumonia caused by A. baumannii 5F1 infection resulted in a survival rate of up to 80 % in mice. In summary, we demonstrated that Perillaldehyde is promising as a new drug for the treatment of pneumonia caused by A. baumannii 5F1 infection.

In situ delivery of a curcumin-loaded dynamic hydrogel for the treatment of chronic peripheral neuropathy.

Patients with peripheral nerve injuries would highly likely suffer from chronic neuropathic pain even after surgical intervention. The primary reasons for this involve sustained neuroinflammatory and dysfunctional changes in the nervous system after the nerve injury. We previously reported an injectable boronic ester-based hydrogel with inherent antioxidative and nerve protective properties. Herein, we first explored the anti-neuroinflammatory effects of Curcumin on primary sensory neurons and activated macrophages in vitro. Next, we incorporated thiolated Curcumin-Pluronic F-127 micelles (Cur-M) into our boronic ester-based hydrogel to develop an injectable hydrogel that serves as sustained curcumin release system (Gel-Cur-M). By orthotopically injecting the Gel-Cur-M to sciatic nerves of mice with chronic constriction injuries, we found that the bioactive components could remain on the nerves for at least 21 days. In addition, the Gel-Cur-M exhibited superior functions compared to Gel and Cur-M alone, which includes ameliorating hyperalgesia while simultaneously improving locomotor and muscular functions after the nerve injury. This could stem from in situ anti-inflammation, antioxidation, and nerve protection. Furthermore, the Gel-Cur-M also showed extended beneficial effects for preventing the overexpression of TRPV1 as well as microglial activation in the lumbar dorsal root ganglion and spinal cord, respectively, which also contributed to its analgesic effects. The underlying mechanism may involve the suppression of CC chemokine ligand-2 and colony-stimulating factor-1 in the injured sensory neurons. Overall, this study suggests that orthotopic injection of the Gel-Cur-M is a promising therapeutic strategy that especially benefits patients with peripheral neuropathy who require surgical interventions.

Genetically encoded sensors for measuring histamine release both in vitro and in vivo.

Histamine (HA) is a key biogenic monoamine involved in a wide range of physiological and pathological processes in both the central and peripheral nervous systems. Because the ability to directly measure extracellular HA in real time will provide important insights into the functional role of HA in complex circuits under a variety of conditions, we developed a series of genetically encoded G-protein-coupled receptor-activation-based (GRAB) HA (GRABHA) sensors with good photostability, sub-second kinetics, nanomolar affinity, and high specificity. Using these GRABHA sensors, we measured electrical-stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. Moreover, we recorded HA release in the preoptic area of the hypothalamus and prefrontal cortex during the sleep-wake cycle in freely moving mice, finding distinct patterns of HA dynamics between these specific brain regions. Thus, GRABHA sensors are robust tools for measuring extracellular HA transmission in both physiological and pathological processes.

Dorsal raphe serotonergic neurons suppress feeding through redundant forebrain circuits.

OBJECTIVE: Serotonin (5HT) is a well-known anorexigenic molecule, and 5HT neurons of dorsal raphe nucleus (DRN) have been implicated in suppression of feeding; however, the downstream circuitry is poorly understood. Here we explored major projections of DRN5HT neurons for their capacity to modulate feeding. METHODS: We used optogenetics to selectively activate DRN5HT axonal projections in hypothalamic and extrahypothalamic areas and monitored food intake. We next used fiber photometry to image the activity dynamics of DRN5HT axons and 5HT levels in projection areas in response feeding and metabolic hormones. Finally, we used electrophysiology to determine how DRN5HT axons affect downstream neuron activity. RESULTS: We found that selective activation of DRN5HT axons in (DRN5HT → LH) and (DRN5HT → BNST) suppresses feeding whereas activating medial hypothalamic projections has no effect. Using in vivo imaging, we found that food access and satiety hormones activate DRN5HT projections to LH where they also rapidly increase extracellular 5HT levels. Optogenetic mapping revealed that DRN5HT → LHvGAT and DRN5HT → LHvGlut2 connections are primarily inhibitory and excitatory respectively. Further, in addition to its direct action on LH neurons, we found that 5HT suppresses GABA release from presynaptic terminals arriving from AgRP neurons. CONCLUSIONS: These findings define functionally redundant forebrain circuits through which DRN5HT neurons suppress feeding and reveal that these projections can be modulated by metabolic hormones.

Brainstem serotonin neurons selectively gate retinal information flow to thalamus.

Retinal ganglion cell (RGC) types relay parallel streams of visual feature information. We hypothesized that neuromodulators might efficiently control which visual information streams reach the cortex by selectively gating transmission from specific RGC axons in the thalamus. Using fiber photometry recordings, we found that optogenetic stimulation of serotonergic axons in primary visual thalamus of awake mice suppressed ongoing and visually evoked calcium activity and glutamate release from RGC boutons. Two-photon calcium imaging revealed that serotonin axon stimulation suppressed RGC boutons that responded strongly to global changes in luminance more than those responding only to local visual stimuli, while the converse was true for suppression induced by increases in arousal. Converging evidence suggests that differential expression of the 5-HT1B receptor on RGC presynaptic terminals, but not differential density of nearby serotonin axons, may contribute to the selective serotonergic gating of specific visual information streams before they can activate thalamocortical neurons.

The Derived Components of Gnaphalium hypoleucum DC. Reduce Quorum Sensing of Chromobacterium violaceum.

Gnaphalium hypoleucum DC. was first recorded in the Chinese National Pharmacopoeia "Yi Plant Medicine". There is no detailed report on its main components' activity in suppressing the quorum sensing activity (QS) of bacteria. Our study aimed to screen the main components in extracts of G. hypoleucum DC. in order to measure their effects on bacterial QS activity and to explore specific quorum sensing mechanisms that are affected by G. hypoleucum DC. extracts. Crude extracts of G. hypoleucum DC. contained significant amounts of two compounds shown to inhibit bacterial QS activity, namely apigenin and luteolin. Apigenin and luteolin in crude extracts of G. hypoleucum DC. showed substantial inhibition of pigment formation, biofilm production, and motility in Chromobacterium violaceum ATCC 12472 compared to the effects of other phytochemicals from G. hypoleucum DC. Apigenin and luteolin exhibited a strong QS inhibitory effect on C. violaceum, interfering with the violacein pigment biosynthesis by downregulating the vioB, vioC, and vioD genes. In the presence of signal molecules, the QS effect is prevented, and the selected compounds can still inhibit the production of the characteristic purple pigment in C. violaceum. Based on qualitative and quantitative research using genomics and bioinformatics, we concluded that apigenin and luteolin in crude extracts of G. hypoleucum DC can interfere with the generation of QS in C. violaceum by downregulating the vioB, vioC, and vioD genes. Indeed, G. hypoleucum DC. is used for the treatment of bacterial infections, and this research provides new ideas and potential alternative uses for medicinal plants.

Construction and Validation of Prediction Model of Severe Abdominal Pain Post-Transarterial Chemoembolization in Patients with HBV-Associated Primary Liver Cancer.

Objective: This study is aimed at constructing and evaluating a prediction model of severe abdominal pain post-transcatheter arterial chemoembolization in patients with HBV-related primary liver cancer. Methods: Patients with HBV-associated primary liver cancer who received transarterial chemoembolization (TACE) from March 2019 to March 2022 in the Interventional Therapy Department of our hospital were selected as the subjects, and the included 160 patients were randomly divided into modeling group (n = 120) and validation group (n = 40) in a ratio of 3 : 1. Visual analog scale (VAS) was used to assess pain severity. 120 patients in the modeling group were divided into no/mild abdominal pain group and severe abdominal pain group. The clinical data of the patients, including gender, age, TACE treatment history, vascular invasion, maximum diameter of tumor, infarction degree, preoperative Eastern Oncology Collaboration Group (ECOG) score, and Lipiodol dosage, were analyzed. Receiver operating characteristic (ROC) curve was used to evaluate the predictive value of the prediction model for severe abdominal pain post-TACE. Results: A total of 116 patients (72.50%) had severe abdominal pain after TACE. Univariate analysis showed that severe abdominal pain after TACE in the modeling group was associated with TACE treatment history, maximum tumor diameter, infarction degree, and preoperative ECOG score (all P < 0.05), but not related to gender, age, vascular invasion, and Lipiodol dosage (all P > 0.05). Logistic regression analysis showed that TACE treatment history, maximum tumor diameter, infarction degree, and preoperative ECOG score were all independent influencing factors for acute abdominal pain post-TACE in HBV-HCC patients (all P < 0.05). The prediction model equation was Y = -3.673 + 1.722 × TACE treatment history + 1.175 × tumor maximum diameter + 2.064 × infarction degree + 1.555 × preoperative ECOG score. Goodness-of-fit test results showed no significant difference between the established prediction model and the observed value (χ 2 = 1.645, P = 0.560) and R 2 = 0.821, suggesting that the prediction ability of the model was relatively accurate. ROC analysis results showed that the area under the curve (AUC) of severe abdominal pain after TACE was 0.916 (0.862~0.970) and 0.902 (95% CI: 0.841~0.963) in the modeling group and the verification group, respectively. Conclusion: TACE treatment history, tumor maximum diameter, infarction degree, and preoperative ECOG score are independent influencing factors for severe abdominal pain post-TACE in patients with HBV-HCC, and the prediction model established on this basis has good application value.

Berberine-mediated REDD1 down-regulation ameliorates senescence of retinal pigment epithelium by interrupting the ROS-DDR positive feedback loop.

BACKGROUND: Accumulation of age-associated senescent cells accompanied with increased reactive oxygen species (ROS) and inflammatory factors contributes to the progression of age-related macular degeneration (AMD), the main cause of blindness in the elderly. Berberine (BBR) has shown efficacy in the treatment of age-related diseases including diabetes and obesity by decreasing ROS. However, the pharmacological effect of BBR on alleviating retinal aging remains largely unknown. PURPOSE: Our study aimed to investigate the pharmacological effect of BBR as an anti-aging agent in retinal aging and its further molecular mechanisms. METHODS: D-galactose (DG)-induced ARPE-19 cell senescence and retinal aging were employed to evaluate the anti-aging effect of BBR in vivo and in vitro. The siRNA transfection, Western-Blot analyses, SA-ß-Gal assay and immunofluorescence were performed to investigate the potential mechanisms of BBR on anti-aging of RPE. RESULTS: In RPE-choroid of both natural aged and DG-induced accelerated aged mice, oxidative stress was increased along with the up-regulation of p21 expression, which was ameliorated by BBR treatment. BBR down-regulated the expression of REDD1 to decrease intracellular ROS content, attenuating DG-induced senescence in vitro and in vivo. Furthermore, p53 instead of HIF-1α was identified as the transcriptional regulator of REDD1 in DG-induced premature senescence. Importantly, NAC and BBR reversed the expression of p53 and the content of 8-OHdG, indicating that the positive feedback loop of ROS-DNA damage response (DDR) was formed, and BBR interrupted this feedback loop to alleviate DG-induced premature senescence by reducing REDD1 expression. In addition, BBR restored DG-damaged autophagy flux by up-regulating TFEB-mediated lysosomal biosynthesis by inhibiting REDD1 expression, thereby attenuating cellular senescence. CONCLUSION: BBR down-regulates REDD1 expression to interrupt the ROS-DDR positive feedback loop and restore autophagic flux, thereby reducing premature senescence of RPE. Our findings elucidate the promising effects of REDD1 on cellular senescence and the great potential of BBR as a therapeutic approach.

Relationships Between Traditional Chinese Medicine Constitution and Age-Related Cognitive Decline in Chinese Centenarians.

Background: Age-related cognitive decline (ARCD) is a common condition among older adults, affecting 100 million people worldwide. Traditional Chinese Medicine's (TCM) constitution is closely related to the occurrence and development of diseases in the elderly population. However, little is known about the relationships between TCM constitution and ARCD in the super-aged population. The present study aimed to investigate the relationships between the TCM constitution and ARCD in Chinese centenarians and to explore the application of the constitution to prevent ARCD in the elderly population. Methods: Each participant underwent a standardized epidemiological investigation and physical examination, based on the China Hainan Centenarian Cohort Study. Data on the demographic characteristics and TCM constitution were collected using structured questionnaires. Results: The present study included 636 centenarians aged 100-116 years. The prevalence of ARCD was 87.7% (n = 558 centenarians). In multiple linear regression analysis, an inverse relationship between Qi depression and Mini-Mental State Examination scales was significant after controlling for a wide range of other factors (P < 0.05). In multiple logistic regression analysis, Qi depression was positively associated with ARCD after full adjustment (P < 0.05). Conclusion: As the first study in the world, the present study provides strong epidemiological evidence that Qi depression has a significant relationship with ARCD in Chinese centenarians, and regulating Qi depression may be a valuable method to prevent and treat ARCD in the elderly population.

Linkage mapping and QTL analysis of growth traits in Rhopilema esculentum.

R. esculentum is a popular seafood in Asian countries and an economic marine fishery resource in China. However, the genetic linkage map and growth-related molecular markers are still lacking, hindering marker assisted selection (MAS) for genetic improvement of R. esculentum. Therefore, we firstly used 2b-restriction site-associated DNA (2b-RAD) method to sequence 152 R. esculentum specimens and obtained 9100 single nucleotide polymorphism (SNP) markers. A 1456.34 cM linkage map was constructed using 2508 SNP markers with an average interval of 0.58 cM. Then, six quantitative trait loci (QTLs) for umbrella diameter and body weight were detected by QTL analysis based on the new linkage map. The six QTLs are located on four linkage groups (LGs), LG4, LG13, LG14 and LG15, explaining 9.4% to 13.4% of the phenotypic variation. Finally, 27 candidate genes in QTLs regions of LG 14 and 15 were found associated with growth and one gene named RE13670 (sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1-like) may play an important role in controlling the growth of R. esculentum. This study provides valuable information for investigating the growth mechanism and MAS breeding in R. esculentum.

Hyperbaric oxygen therapy does not alleviate tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles.

During tourniquet application, blood flow is restricted to a limb to stop excessive limb hemorrhage in a trauma setting and to create a bloodless operating field in the surgical setting. During tourniquet-related ischemia, aerobic respiration stops, and ATP is depleted, and during subsequent reperfusion, there is an increase in reactive oxygen species (ROS) production and other endogenous substances, which leads to acute ischemia-reperfusion (IR) injuries, including tissue necrosis and skeletal muscle contractile dysfunction. Hyperbaric oxygen (HBO) therapy can increase the arterial oxygen tension in the tissues of patients with general hypoxia/anoxia, including carbon monoxide poisoning, circulatory arrest, and cerebral and myocardial ischemia. Here, we studied the protective effects of HBO pretreatment with 100% oxygen at 2.5 ATA against tourniquet/IR injury in mice. After one hour of HBO therapy with 100% oxygen at 2.5 ATA was administered to C57/BL6 mice, a rubber band was placed at the hip joint of the unilateral hindlimb to induce 3 h of ischemia and then released for 48 h of reperfusion. We analyzed gastrocnemius muscle morphology and contractile function and measured the levels of ATP and ROS accumulation in the muscles. HBO pretreatment did not improve tourniquet/IR-injured gastrocnemius muscle morphology and muscle contraction. Tourniquet/IR mice with HBO pretreatment showed no increase in ATP levels in IR tissues, but they did have a decreased amount of ROS accumulation in the muscles, compared to IR mice with no HBO pretreatment. These data suggest that one hour of HBO pretreatment with 100% oxygen at 2.5 ATA increases the antioxidant response to lower ROS accumulation but does not increase ATP levels in IR muscles and improve tourniquet/IR-injured muscle morphology and contractile function.

Astrocytes Render Memory Flexible by Releasing D-Serine and Regulating NMDA Receptor Tone in the Hippocampus.

BACKGROUND: NMDA receptor (NMDAR) hypofunction has been implicated in several psychiatric disorders with impairment of cognitive flexibility. However, the molecular mechanism of how NMDAR hypofunction with decreased NMDAR tone causes the impairment of cognitive flexibility has been minimally understood. Furthermore, it has been unclear whether hippocampal astrocytes regulate NMDAR tone and cognitive flexibility. METHODS: We employed cell type-specific genetic manipulations, ex vivo electrophysiological recordings, sniffer patch recordings, cutting-edge biosensor for norepinephrine, and behavioral assays to investigate whether astrocytes can regulate NMDAR tone by releasing D-serine and glutamate. Subsequently, we further investigated the role of NMDAR tone in heterosynaptic long-term depression, metaplasticity, and cognitive flexibility. RESULTS: We found that hippocampal astrocytes regulate NMDAR tone via BEST1-mediated corelease of D-serine and glutamate. Best1 knockout mice exhibited reduced NMDAR tone and impairments of homosynaptic and α1 adrenergic receptor-dependent heterosynaptic long-term depression, which leads to defects in metaplasticity and cognitive flexibility. These impairments in Best1 knockout mice can be rescued by hippocampal astrocyte-specific BEST1 expression or enhanced NMDAR tone through D-serine supplement. D-serine injection in Best1 knockout mice during initial learning rescues subsequent reversal learning. CONCLUSIONS: These findings indicate that NMDAR tone during initial learning is important for subsequent learning, and hippocampal NMDAR tone regulated by astrocytic BEST1 is critical for heterosynaptic long-term depression, metaplasticity, and cognitive flexibility.

A Brainstem reticulotegmental neural ensemble drives acoustic startle reflexes.

The reticulotegmental nucleus (RtTg) has long been recognized as a crucial component of brainstem reticular formation (RF). However, the function of RtTg and its related circuits remain elusive. Here, we report a role of the RtTg in startle reflex, a highly conserved innate defensive behaviour. Optogenetic activation of RtTg neurons evokes robust startle responses in mice. The glutamatergic neurons in the RtTg are significantly activated during acoustic startle reflexes (ASR). Chemogenetic inhibition of the RtTg glutamatergic neurons decreases the ASR amplitudes. Viral tracing reveals an ASR neural circuit that the cochlear nucleus carrying auditory information sends direct excitatory innervations to the RtTg glutamatergic neurons, which in turn project to spinal motor neurons. Together, our findings describe a functional role of RtTg and its related neural circuit in startle reflexes, and demonstrate how the RF connects auditory system with motor functions.

RNA demethylation increases the yield and biomass of rice and potato plants in field trials.

RNA N6-methyladenosine (m6A) modifications are essential in plants. Here, we show that transgenic expression of the human RNA demethylase FTO in rice caused a more than threefold increase in grain yield under greenhouse conditions. In field trials, transgenic expression of FTO in rice and potato caused ~50% increases in yield and biomass. We demonstrate that the presence of FTO stimulates root meristem cell proliferation and tiller bud formation and promotes photosynthetic efficiency and drought tolerance but has no effect on mature cell size, shoot meristem cell proliferation, root diameter, plant height or ploidy. FTO mediates substantial m6A demethylation (around 7% of demethylation in poly(A) RNA and around 35% decrease of m6A in non-ribosomal nuclear RNA) in plant RNA, inducing chromatin openness and transcriptional activation. Therefore, modulation of plant RNA m6A methylation is a promising strategy to dramatically improve plant growth and crop yield.

The effect of Chinese herbal medicine on digestive system and liver functions should not be neglected in COVID-19: An updated systematic review and meta-analysis.

Gastrointestinal symptoms and liver injury are common in patients with coronavirus disease 2019 (COVID-19). However, profiles of different pharmaceutical interventions used are relatively underexplored. Chinese herbal medicine (CHM) has been increasingly used for patients with COVID-19, but the efficacy of CHM used in COVID-19 on gastrointestinal symptoms and liver functions has not been well studied with definitive results based on the updated studies. The present study aimed at testing the efficacy of CHM on digestive symptoms and liver function (primary outcomes), the aggravation of COVID-19, and the time to viral assay conversion (secondary outcomes), among patients with COVID-19, compared with standard pharmacotherapy. The literature search was undertaken in 11 electronic databases from December 1, 2019 up to November 8, 2020. Appraisal of the evidence was conducted with Cochrane risk of bias tool or Newcastle Ottawa Scale. A random-effects model or subgroup analysis was conducted when significant heterogeneity was identified in the meta-analysis. The certainty of the evidence was assessed with the grading of recommendations assessment, development, and evaluation approach. Forty-eight included trials involving 4,704 participants were included. Meta-analyses favored CHM plus standard pharmacotherapy for COVID-19 on reducing the aggravation of COVID-19 and the time to viral assay conversion compared with standard pharmacotherapy. However, the present CHM as a complementary therapy for treating COVID-19 may not be beneficial for improving most gastrointestinal symptoms and liver function based on the current evidence. More well-conducted trials are warranted to confirm the potential efficacy of CHM furtherly.

Astrocytes Control Sensory Acuity via Tonic Inhibition in the Thalamus.

Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release.

Pitfalls of using denosumab preoperatively to treat refractory severe hypercalcaemia.

A 40-year-old man, with a history of metastatic parathyroid carcinoma, status post primary tumour resection and lung metastasectomy, was hospitalised for persistent severe hypercalcaemia and elevated parathyroid hormone levels despite conventional management and escalating doses of cinacalcet. A single dose (120 mg) of denosumab was given and his calcium level plummeted from 14.8 mg/dL to 5.5 mg/dL. After second lung metastasectomy, he developed prolonged hypocalcaemia that required calcium and vitamin D supplements for more than 3 years. In patients with severe hypercalcaemia refractory to conventional therapies, denosumab has been used off-label with some success. A known side effect of denosumab is hypocalcaemia, which is often short-lived. The risk of prolonged hypocalcaemia should be fully evaluated before using denosumab preoperatively, especially in patients with renal insufficiency, prolonged hyperparathyroidism or anticipated tumour debulking surgery.

Paternal chronic folate supplementation induced the transgenerational inheritance of acquired developmental and metabolic changes in chickens.

Increasing evidence indicates that paternal diet can result in metabolic changes in offspring, but the definite mechanism remains unclear in birds. Here, we fed breeder cocks five different diets containing 0, 0.25, 1.25, 2.50 and 5.00 mg kg-1 folate throughout life. Paternal folate supplementation (FS) was beneficial to the growth and organ development of broiler offspring. Most importantly, the lipid and glucose metabolism of breeder cocks and broiler offspring were affected by paternal FS, according to biochemical and metabolomic analyses. We further employed global analyses of hepatic and spermatozoal messenger RNA (mRNA), long non-coding RNA (lncRNA) and micro RNA (miRNA). Some key genes involved in the glycolysis or gluconeogenesis pathway and the PPAR signalling pathway, including PEPCK, ANGPTL4 and THRSP, were regulated by differentially expressed hepatic and spermatozoal miRNAs and lncRNAs in breeder cocks and broiler offspring. Moreover, the expression of ANGPTL4 could also be regulated by differentially expressed miRNAs and lncRNAs in spermatozoa via competitive endogenous RNA (ceRNA) mechanisms. Overall, this model suggests that paternal folate could transgenerationally regulate lipid and glucose metabolism in broiler offspring and the epigenetic transmission may involve altered spermatozoal miRNAs and lncRNAs.

Spermatozoal mRNAs expression implicated in embryonic development were influenced by dietary folate supplementation of breeder roosters by altering spermatozoal piRNA expression profiles.

Dietary folate intake, together with changes in its metabolism process, have effects on male reproduction, sperm epigenetic patterning and offspring outcome. Previous studies have proven that PIWI-interacting RNAs (piRNAs) play important roles in successful spermatogenesis and regulating genes expression of sperm and offspring embryo. Herein, we fed breeder roosters with five different levels (0, 0.25, 1.25, 2.50, and 5.00 mg/kg) of folate throughout life and found that paternal folate supplementation was beneficial to the growth and organ development of offspring broilers. Further spermatozoal mRNAs sequencing analyses implied that the dietary folate supplementation could regulate the spermatozoal mRNA abundance of genes related to the fetal development. Furthermore, global piRNAs analyses of breeder roosters' sperm revealed that differential concentration of dietary folate supplementation could change piRNAs profiles. Combined mRNAs sequencing and target gene prediction of differentially expressed gene-derived piRNAs, embryonic development and metabolism related pathways and biological processes, which were consisted to the regulatory roles of paternal folate supplementations, were significantly affected by the differentially expressed gene-derived piRNAs based on the GO and KEGG analyses. Overall, our results provided a novel insight into the role of piRNAs in response to folate intake, which will broaden the understanding about the relationship between folate and sperm epigenetic patterning of breeder roosters.