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Abstract This study aimed to evaluate the impact of prolonged fasting on the physiological and biochemical alterations and metabolic shifts in healthy adults and to provide experimental data and theoretical support for the hypometabolic state induced by prolonged fasting. Thirteen volunteers were selected through public recruitment to undergo a 21-day complete fasting experiment. The experimental period lasted 34 days, including a 3-day baseline, 21-day completing fasting, 5-day calorie restriction and 5-day full recovery diet. Physiological indicators such as body weight, blood pressure, blood glucose, blood ketones, and blood uric acid were evaluated along with resting metabolic rate, routine blood tests, liver function, and heart function indexes employing traditional approaches. During the 21-day complete fasting period, there was a significant decrease in body weight (average − 14.96 ± 1.55%), a reduction in blood glucose (average − 21.63 ± 0.058%), an increase in blood ketones (from baseline 0.1 ± 0.04 mmol/L to 6.61 ± 1.25 mmol/L) and blood uric acid (from baseline 385.38 ± 57.78 µmol/L to 866.31 ± 172.01 µmol/L), a continuous decline in resting energy expenditure (average − 20.3 ± 11.13%), and the respiratory quotient tending towards fat metabolism. Most of the items in the complete blood count and liver indicators remained stable and within the normal range. Heart function showed functional adaptive changes without structural damage. Prolonged fasting can reduce the body’s resting energy expenditure and adapt to body weight loss through physiological regulatory mechanisms without adverse effects on basic physiological functions or the structure of important organs. Under medical supervision, healthy adults can safely engage in prolonged fasting for up to 21 days with metabolic adaption and no damage to pivotal organ, which could provide potential technical support for human health and survival strategies in extreme conditions such as food shortages during long-duration manned spaceflight.

Hibernation, an adaptive mechanism to extreme environmental conditions, is prevalent among mammals. Its main characteristics include reduced body temperature and metabolic rate. However, the mechanisms by which hibernating animals re-enter deep sleep during the euthermic phase to sustain hibernation remain poorly understood. We selected the Tamias sibiricus as a model organism and conducted transcriptomic sequencing of its hypothalamus at multiple time points throughout hibernation. Through the strategies of gene set filtering and intersection analysis, we effectively filtered out redundant data, identifying a subset of genes whose expression was downregulated during the euthermic phase potentially inducing re-enter deep sleep, thereby maintaining the periodic cycles of torpor and arousal. These cycles are crucial for sustaining the overall hibernation process. Notably, genes associated with sodium and potassium ion channels were significantly enriched. Specifically, potassium ion-related genes such as Kcnc1, Kcna2, Kcng4, and Kcna6, along with sodium ion-related genes such as Scn1a and Hcn2, were markedly downregulated. qRT-PCR validation of four of these genes (Kcnc1, Kcna6, Scn1a, and Hcn2) confirmed significant downregulation during the euthermic phase compared to the deep sleep phase, further supporting our transcriptomic findings. This study provides novel insights into the hypothalamic transcriptome dynamics at various hibernation stages. Although the functional roles of these genes require further investigation, our findings lay the groundwork for future studies to elucidate the molecular mechanisms underlying hibernation.

The hypometabolism induced by fasting has great potential in maintaining health and improving survival in extreme environments, among which thyroid hormone (TH) plays an important role in the adaptation and the formation of new energy metabolism homeostasis during long-term fasting. In the present review, we emphasize the potential of long-term fasting to improve physical health and emergency rescue in extreme environments, introduce the concept and pattern of fasting and its impact on the body’s energy metabolism consumption. Prolonged fasting has more application potential in emergency rescue in special environments. The changes of THs caused by fasting, including serum biochemical characteristics, responsiveness of the peripheral and central hypothalamus-pituitary-thyroid (HPT) axis, and differential changes of TH metabolism, are emphasized in particular. It was proposed that the variability between brain and liver tissues in THs uptake, deiodination activation and inactivation is the key regulatory mechanism for the cause of peripheral THs decline and central homeostasis. While hypothalamic tanycytes play a pivotal role in the fine regulation of the HPT negative feedback regulation during long-term fasting. The study progress of tanycytes on thyrotropin-releasing hormone (TRH) release and deiodination is described in detail. In conclusion, the combination of the decrease of TH metabolism in peripheral tissues and stability in the central HPT axis maintains the basal physiological requirement and new energy metabolism homeostasis to adapt to long-term food scarcity. The molecular mechanisms of this localized and differential regulation will be a key research direction for developing measures for hypometabolic applications in extreme environment.

Osteocalcin (Ocn), also known as bone Gla protein, is synthesized by osteoblasts and thought to regulate energy metabolism, testosterone synthesis and brain development. However, its function in bone is not fully understood. Mice have three Ocn genes: Bglap, Bglap2 and Bglap3. Due to the long span of these genes in the mouse genome and the low expression of Bglap3 in bone, researchers commonly use Bglap and Bglap2 knockout mice to investigate the function of Ocn. However, it is unclear whether Bglap3 has any compensatory mechanisms when Bglap and Bglap2 are knocked out. Considering the controversy surrounding the role of Ocn in bone, we constructed an Ocn-deficient mouse model by knocking out all three genes (Ocn−/−) and analyzed bone quality by Raman spectroscopy (RS), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and MicroCT (μCT). The RS test showed that the alignment of hydroxyapatite crystals and collagen fibers was significantly poorer in Ocn−/− mice than in wild-type (WT) mice. Ocn deficiency resulted in a looser surface structure of bone particles and a larger gap area proportion. FTIR analysis showed few differences in bone mineral index between WT and Ocn−/− mice, while μCT analysis showed no significant difference in cortical and trabecular regions. However, under tail-suspension simulating bone loss condition, the disorder of hydroxyapatite and collagen fiber alignment in Ocn−/− mice led to more obvious changes in bone mineral composition. Collectively, our results revealed that Ocn is necessary for regulating the alignment of minerals parallel to collagen fibrils.

Background Fasting has been widely used to improve various metabolic diseases in humans. Adaptive fasting is necessary for metabolic adaptation during prolonged fasting, which could overcome the great advantages of short-term fasting. The liver is the main organ responsible for energy metabolism and metabolic homeostasis. To date, we lack literature that describes the physiologically relevant adaptations of the liver during prolonged fasting and refeeding. For that reason, this study aims to evaluate the response of the liver of Sprague-Dawley (SD) rats to prolonged fasting and refeeding. Methods Sixty-six male SD rats were divided into the fasting groups, which were fasted for 0, 4, 8, 12, 24, 48, 72, or 96 h, and the refeeding groups, which were refed for 1, 3, or 6 days after 96 h of fasting. Serum glucose, TG, FFA, β-hydroxybutyrate, insulin, glucagon, leptin, adiponectin and FGF21 levels were assessed. The glucose content, PEPCK activity, TG concentration and FFA content were measured in liver tissue, and the expression of genes involved in gluconeogenesis (PEPCK and G6Pase), ketogenesis (PPARα, CPT-1a and HMGCS2) and the protein expression of nutrient-sensing signaling molecules (AMPK, mTOR and SIRT1) were determined by RT-qPCR and western blotting, respectively. Results Fasting significantly decreased the body weight, which was totally recovered to baseline after 3 days of refeeding. A 4-day fast triggered an energy metabolic substrate shift from glucose to ketones and caused serum hormone changes and changes in the protein expression levels of nutrient-sensing signaling molecules. Glycogenolysis served as the primary fuel source during the first 24 h of fasting, while gluconeogenesis supplied the most glucose thereafter. Serum FFA concentrations increased significantly with 48 h of fasting. Serum FFAs partly caused high serum β-hydroxybutyrate levels, which became an important energy source with the prolongation of the fasting duration. One day of refeeding quickly reversed the energy substrate switch. Nutrient-sensing signaling molecules (AMPK and SIRT1 but not mTOR signaling) were highly expressed at the beginning of fasting (in the first 4 h). Serum insulin and leptin decreased with fasting initiation, and serum glucagon increased, but adiponectin and FGF21 showed no significant changes. Herein, we depicted in detail the timing of the metabolic response and adaptation of the liver to a 4-day water-only fast and subsequent refeeding in rats, which provides helpful support for the design of safe prolonged and intermittent fasting regimens.

Osteocalcin (OCN) was the most abundant noncollagen protein and considered as an endocrine factor. However, the functions of Undercarboxylated osteocalcin (ucOCN) on osteoclast and bone resorption are not well understood. In the present study, preosteoclast RAW264.7 cells and bone marrow mononuclear cells (BMMs) were treated with ucOCN purified from prokaryotic bacteria. Our results showed that ucOCN attenuated the proliferation of RAW264.7 cells with a concentration dependant manner by MTS assay. Scrape wounding assay revealed the decreased motility of RAW264.7 cells after ucOCN treatment. RT-qPCR results manifested the inhibitory effects of ucOCN on the expression of osteoclastic marker genes in RAW264.7 cells during inducing differentiation of RANKL. It was also observed that ucOCN inhibited the formation of multinucleated cells from RAW264.7 cells and BMMs detected by TRAP staining. The number and area of bone resorb pits were also decreased after treatment with ucOCN during their osteoclast induction by toluidine blue staining. The formation and integrity of the osteoclast actin ring were impaired by ucOCN by immunofluorescent staining. Time dependant treatment of ucOCN during osteoclastic induction demonstrated the inhibitory effects mainly occurred at the early stage of osteoclastogenesis. Signaling analysis of luciferase activity of the CRE or SRE reporter and ERK1/2 phosphorylation showed the selective inhibitor or siRNA of Gprc6a (a presumptive ucOCN receptor) could attenuate the promotion of ucOCN on CRE-luciferase activity. Taken together, we provided the first evidence that ucOCN had negative effects on the early differentiation and bone resorption of osteoclasts via Gprc6a.

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis. During early-stage infection, M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected via nested-polymerase chain reaction (PCR) experiments. Little research has focused on immune responses in nested PCR-positive (bTB PCR-P) or nested PCR-negative (bTB PCR-N) M. bovis-infected cattle. Here, we investigated the transcriptomes of peripheral blood mononuclear cells (PBMCs), with or without stimulation by purified protein derivative of bovine tuberculin (PPD-B), among bTB PCR-P, bTB PCR-N, and healthy cattle using RNA-Seq. We also explored the potential value of PBMC transcripts as novel biomarkers for diagnosing bTB. Numerous differentially expressed genes were identified following pair-wise comparison of different groups, with or without PPD-B stimulation (adjusted p < 0.05). Compared with healthy cattle, bTB PCR-P, and bTB PCR-N cattle shared 5 significantly dysregulated biological pathways, including Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, Hematopoietic cell lineage, Osteoclast differentiation and HTLV-I infection. Notably, dysregulated biological pathways of bTB PCR-P and bTB PCR-N cattle were associated with cell death and phagocytosis, respectively. Lymphotoxin alpha and interleukin-8 could potentially differentiate M. bovis-infected and healthy cattle upon stimulation with PPD-B, with area-under-the-curve (AUC) values of 0.9991 and 0.9343, respectively. B cell lymphoma 2 and chitinase 3-like 1 might enable differentiation between bTB PCR-P and bTB PCR-N upon stimulation with PPD-B, with AUC values of 0.9100 and 0.8893, respectively. Thus, the PBMC transcriptome revealed the immune responses in M. bovis-infected cattle (bTB PCR-P and bTB PCR-N) and may provide a novel sight in bTB diagnosis.

Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.

Two strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in 2006 and 2016 and designated as FZ06A and FZ16A, respectively. Inoculation experiments showed that FZ06A caused 100% morbidity and 60% mortality, while FZ16A caused 100% morbidity without death. By using genomic sequence and phylogenetic analyses, close relationships between a Chinese highly pathogenic PRRSV strain and the FZ06A and FZ16A strains were observed. Based on the achieved results, multiple genomic variations in Nsp2, a unique N-glycosylation site (N33→K33), and a K151 amino acid (AA) substitution for virulence in the GP5 of FZ16A were detected; except the 30 AA deletion in the Nsp2-coding region. Inoculation experiments were conducted and weaker virulence of FZ16A than FZ06A was observed. Based on our results, a 30 AA deletion in the Nsp2-coding region is an unreliable genomic indicator of a high virulence PRRSV strain. The Nsp2 and GP5 differences, in addition to the virulence difference between these two highly pathogenic PRRSV strains, have the potential to be used to establish a basis for further study of PRRSV virulence determinants and to provide data useful in the development of vaccines against this economically devastating disease. [ABSTRACT FROM AUTHOR]