MiRNA-seq and mRNA-seq revealed the mechanism of fluoride-induced cauda epididymal injury.

The widespread presence of fluoride in water, food, and the environment continues to exacerbate the impact of fluoride on the male reproductive health. However, as a critical component of the male reproductive system, the intrinsic mechanism of fluoride-induced cauda epididymis damage and the role of miRNAs in this process are still unclear. This study established a mouse fluorosis model and employed miRNA and mRNA sequencing; Evans blue staining, Oil Red O staining, TEM, immunofluorescence, western blotting, and other technologies to investigate the mechanism of miRNA in fluoride-induced cauda epididymal damage. The results showed that fluoride exposure increased the fluoride concentration in the hard tissue and cauda epididymis, altered the morphology and ultrastructure of the cauda epididymis, and reduced the motility rate, normal morphology rate, and hypo-osmotic swelling index of the sperm in the cauda epididymis. Furthermore, sequencing results revealed that fluoride exposure resulted in differential expression of 17 miRNAs and 4725 mRNAs, which were primarily enriched in the biological processes of tight junctions, inflammatory response, and lipid metabolism, with miR-742-3p, miR-141-5p, miR-878-3p, and miR-143-5p serving as key regulators. Further verification found that fluoride damaged tight junctions, raised oxidative stress, induced an inflammatory response, increased lipid synthesis, and reduced lipid decomposition and transport in the cauda epididymis. This study provided a theoretical basis for developing miRNA as potential diagnostic markers and therapeutic target drugs for this injury.

Exercise Alleviates Fluoride-Induced Learning and Memory Impairment in Mice: Role of miR-206-3p and PREG.

Fluorosis decreases the learning and memory ability in humans and animals, while exercise can reduce the risk of cognitive decline. However, the effect of exercise on learning and memory in fluoride-exposed mice is unclear. For this purpose, in this study, mice were randomly allotted into four groups (16 mice per group, half male and half female): control group (group C), fluoride group (group F, 100 mg/L sodium fluoride (NaF)), exercise group (group E, treadmill exercise), and E plus F group (group EF, treadmill exercise, and 100 mg/L NaF). During 6 months of exposure, exercise alleviated the NaF-induced decline in memory and learning. In addition, NaF induced injuries in mitochondria and myelin sheath ultrastructure and reduced the neurons number, while exercise restored them. Metabolomics results showed that phosphatidylethanolamine, pregnenolone (PREG), and lysophosphatidic acid (LysoPA) were altered among groups C, F, and EF. Combined with previous studies, it can be suggested that PREG might be a biomarker in response to exercise-relieving fluorine neurotoxicity. The miRNA sequencing results indicated that in the differently expressed miRNAs (DEmiRNAs), miR-206-3p, miR-96-5p, and miR-144-3p were shared in groups C, F, and EF. After the QRT-PCR validation and in vitro experiments, it was proved that miR-206-3p could reduce cell death and regulate AP-1 transcription factor subunit (JunD) and histone deacetylase 4 (HDAC4) to alleviate fluoride neurotoxicity. To sum up, the current study reveals that exercise could alleviate NaF-induced neurotoxicity by targeting miR-206-3p or PREG, which will contribute to revealing the pathogenesis and therapeutic method of fluoride neurotoxicity.

α-Lipoic acid improves mitochondrial biogenesis and dynamics by enhancing antioxidant and inhibiting Wnt/Ca2+ pathway to relieve fluoride-induced hepatotoxic injury.

Fluoride (F), widely present in water and food, poses a serious threat to liver health, and oxidative damage and mitochondrial damage are its main causes. As a natural mitochondrial protector and antioxidant, α-lipoic acid (ALA)'s alleviating effect on fluorosis liver injury and its underlying mechanism are still unclear. Therefore, this study established a fluorosis ALA intervention mice model to explore the mechanism of mitochondrial biogenesis, mitochondrial dynamics, and Wnt/Ca2+ pathway in ALA attenuating fluorosis liver injury. The results showed that ALA mitigated F-induced weight loss, hepatic structural and functional damage, hepatocytes mitochondrial damage, and decreased antioxidant levels. However, ALA did not reduce F accumulation in the femur. Further mRNA and protein detection results showed that F increased the expression levels of key genes in the mitochondrial fission (Drp1, Mff, and Fis1), mitophagy (Parkin, Pink1, and Prdx3), Wnt/Ca2+ pathway (Wnt5a and CaMK2), and rised the number and intensity of fluorescent spots of Drp1, but decreased the expression levels of key genes in the mitochondrial biogenesis (Sirt1, Sirt3, and PGC-1α) and fusion (OPA1, Mfn2, and Mfn1), and reduced the number and intensity of fluorescent spots of PGC-1α in the liver. However, the intervention of ALA relieved the F-induced changes in the expressions of the above genes. In conclusion, ALA mitigated F-induced hepatic injury through enhancing antioxidant capacity and inhibiting Wnt/Ca2+ pathway to improve mitochondrial biogenesis and dynamics disturbance. This study further reveals the hepatotoxic mechanism of F and the protective mechanism of ALA, and provides a theoretical basis for ALA as a potential preventive and palliative agent for F-induced hepatotoxic injury.

Effects of Treadmill Exercise on Liver Apoptosis in Fluoride-Exposed Mice.

Hepatotoxicity induced by excessive fluoride (F) exposure has been extensively studied in both humans and animals. Chronic fluorosis can result in liver apoptosis. Meanwhile, moderate exercise alleviates apoptosis caused by pathological factors. However, the effect of moderate exercise on F-induced liver apoptosis remains unclear. In this research, sixty-four three-week-old Institute of Cancer Research (ICR) mice, half male and half female, were randomly divided into four groups: control group (distilled water); exercise group (distilled water and treadmill exercise); F group [100 mg/L sodium fluoride (NaF)]; and exercise plus F group (100 mg/L NaF and treadmill exercise). The liver tissues of mice were taken at 3 months and 6 months, respectively. Hematoxylin-eosin (HE) staining and situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) results showed that nuclear condensation and apoptotic hepatocytes occurred in the F group. However, this phenomenon could be reversed with the intervention of treadmill exercise. The results of QRT-PCR and western blot displayed NaF- induced apoptosis via tumor necrosis factor recpter 1 (TNFR1) signaling pathway, while treadmill exercise could restore the molecular changes caused by excessive NaF exposure.

Multiomics Analysis Revealed the Molecular Mechanism of miRNAs in Fluoride-Induced Hepatic Glucose and Lipid Metabolism Disorders.

Fluoride-induced liver injury seriously endangers human and animal health and animal food safety, but the underlying mechanism remains unclear. This study aims to explore the mechanism of miRNAs in fluoride-induced hepatic glycolipid metabolism disorders. C57 male mice were used to establish the fluorosis model (22.62 mg/L F-, 12 weeks). The results indicated that fluoride increased fluoride levels, impaired the structure and function, and disrupted the glycolipid metabolism in the liver. Furthermore, the sequencing results showed that fluoride exposure resulted in the differential expression of 35 miRNAs and 480 mRNAs, of which 23 miRNAs were related to glycolipid metabolism. miRNA-mRNA network analyses and RT-PCR revealed that miRNAs mediated fluoride-induced disturbances in the hepatic glycolipid metabolism. Its possible mechanism was to regulate the insulin pathway, PPAR pathway, and FOXO pathway, which in turn affected the bile secretion, the metabolic processes of glucose, the decomposition of lipids, and the synthesis of unsaturated fatty acids in the liver. This study provides a theoretical basis for miRNAs as diagnostic indicators and target drugs for the treatment of fluoride-induced liver injury.

Moderate exercise relieves fluoride-induced liver and kidney inflammatory responses through the IKKß/NFκB pathway.

With the intensification of environmental pollution, the content of fluoride is increasing in human and animal living environments. Long-term fluoride exposure can cause damage to the liver and kidney, which are the main sites for fluoride metabolism, storage and removal. Moreover, exercise often accompanies the entire process of fluoride exposure in humans and animals. However, the mechanism of exercise on fluoride-induced liver and kidney injury remains unclear. Hence, we established a fluoride exposure and/or exercise mouse model to explore the influence of exercise on fluoride-induced liver and kidney inflammation and the potential mechanism. The results showed that fluoride caused obvious structural and functional damage and the notable recruitment of immunocytes in the liver and kidney. In addition, fluoride increased the levels of IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-21, TNF-α, and TGF-ß but decreased the ratio of IFN-γ/IL-4 and IL-2/IL-10, which indicated that fluoride disturbed the inflammatory balance and caused hepatonephritis. In addition, the expression levels of IKKß and NFκB were increased, and the expression of IκBα was decreased after fluoride exposure, indicating that fluoride activated the IKKß/NFκB pathway. In summary, long-term moderate treadmill exercise relieved fluoride-induced liver and kidney inflammatory responses through the IKKß/NFκB pathway, and exercise can be used to prevent fluoride-induced liver and kidney damage.

OsciDrop: A Versatile Deterministic Droplet Generator.

This paper describes OsciDrop, a versatile chip-free droplet generator used to produce size-tunable droplets on demand. Droplet generation is fundamental to miniaturized analysis. We designed OsciDrop to segment the fluid flowing out of the orifice of a disposable pipette tip into droplets by oscillating its distal end underneath an immiscible continuous phase. We described the theoretical model and investigated the effect of flow rate, oscillating amplitude, frequency, and waveform on droplet generation. Our study revealed a previously underexplored Weber number-dominated regime that leverages inertial force instead of viscous force to generate droplets. The same pipette tip allowed robust and deterministic generation of monodisperse droplets with programmable sizes ranging from 200 pL to 2 µL by asymmetrical oscillation. We validated this platform with two droplet-based nucleic acid amplification tests: a digital loop-mediated isothermal amplification assay for absolute quantification of African swine fever virus and a multi-volume digital polymerase chain reaction assay for the high dynamic range measurement of human genomic DNA. The OsciDrop method opens a facile avenue to miniaturization, integration, and automation, exhibiting full accessibility for digital molecular diagnostics.

Alleviative Effects of Exercise on Bone Remodeling in Fluorosis Mice.

Fluorine is widely present in nature in the form of fluoride. Prolonged high-dose fluoride exposure can cause skeletal fluorosis, resulting in osteosclerosis, osteoporosis or osteomalacia. It has been proved that exercise is one of the important factors affecting the health of the bone and promoting bone formation. To investigate the effects of exercise on bone remodeling in fluorosis mice, 120 male 3-week-old ICR mice were randomly divided into four groups: control group (C), exercise group (E), fluoride group (F), fluoride plus exercise group (F + E). After 8-week physical exercise and/or fluoride exposure, we evaluated the content of fluorine, the histopathological structure and microstructure of femur, bone metabolism biochemical indexes and oxidative stress related parameters, and the mRNA and protein levels of genes in BMP-2/Smads and OPG/RANKL/RANK signaling pathways. Our results showed that 100 mg/L NaF exposure increased the accumulation of fluoride in bone, altered histology of bone, and enhanced the activities of ALP and TRACP. Meanwhile, excessive fluoride induced oxidative stress in bone tissue by increasing the content of ROS and MDA, and decreasing the activities of antioxidant enzymes. In addition, the results of qRT-PCR suggested that NaF significantly increased the mRNA expression of BMP-2, Smad-5, Col IA1, Col IA2, OPG, RANKL and RANK, as well as the elevated proteins of OPG, RANKL and RANK. However, these fluoride-induced changes were alleviated after moderate exercise. Taken together, these findings indicated that moderate exercise decreased the toxicity of fluoride by reducing the accumulation of fluorine in the body to relieve the bone damage caused by fluorosis.

Exercise Ameliorates Fluoride-induced Anxiety- and Depression-like Behavior in Mice: Role of GABA.

Fluoride exposure caused anxiety- and depression-like behavior in mice. Meanwhile, exercise contributes to relieve anxiety and depression. However, the effects of exercise on anxiety- and depression-like behavior in fluorosis mice remain unclear. In the current study, thirty-six Institute of Cancer Research (ICR) female mice were randomly assigned to four groups: control group (C, gavage with distilled water); exercise group (E, gavage with distilled water and treadmill exercise (speed, 10 m/min; time, 30 min/day)); fluoride group (F, gavage with 24 mg/kg sodium fluoride (NaF)); and exercise plus fluoride group (EF, gavage with 24 mg/kg NaF and treadmill exercise). All treatments lasted for 8 weeks. A number of entries into and time spent in the open zone in the elevated zero maze (EZM), resting time in the tail suspension test (TST) and levels of serotonin (5-HT) and gamma-aminobutyric acid (GABA), were significantly altered in F when compared to C. Meanwhile, the anxiety-like behavior in the EZM and the depression-like behavior in the TST were significantly improved in EF when compared to group F. Exercise significantly enhanced fluoride-induced low GABA level, with less effect on the concentration of 5-HT. Moreover, the mRNA and protein expressions of GABA synthesis and transport-related proteins of glutamic acid decarboxylase (GAD) 65 and GAD67 and vesicular GABA transporter (VGAT) were all strikingly decreased in F, while those in EF were increased. In conclusion, exercise ameliorates anxiety- and depression-like behavior in fluorosis mice through increasing the expressions of GABA synthesis and transport-related proteins, rather than 5-HT system.

Exercise alleviated intestinal damage and microbial disturbances in mice exposed to fluoride.

Gastrointestinal reaction is an important symptom of fluorosis and is associated with intestinal morphological and functional impairment. Regular moderate exercise may reduce the incidence of infection and contribute to the maintenance of intestinal mucosal function and immune homeostasis. In this study, the mice were randomly divided to four groups: control group (C, distilled water), exercise group (E, distilled water and treadmill exercise), fluoride group (F, 100 mg/L NaF), and exercise plus fluoride group (EF, 100 mg/L NaF and treadmill exercise). The treadmill exercise was performed as 5 m/min, 5 min; 10 or 12 m/min, 20 min; 5 m/min, 5 min, with 5 consecutive days per week. After 6 months, exercise alleviated the intestinal morphological structure damage and restored the villus height (VH) and VH/crypt depth (VH/CD) in the duodenum of fluoride-exposed mice. Exercise decreased the mRNA expressions of IL-1ß, IL-6, TNF-α, TLR2 and NF-κB (p65) in fluoride-exposed mice, and restored the gene levels of Occludin and ZO-1 in the duodenum, as well as Occludin, ZO-1, and Claudin-1 in the colon. Although there were no significant differences in the Occludin and ZO-1 protein expressions between F and EF, two proteins in EF presented statistical homogeneousness when compared with the C. The 16S rDNA high-throughput sequencing found that exercise restored the variations in intestinal microbiota composition and the abundances of specific bacteria in fluoride-exposed mice, including increasing the abundances of Epsilonbacteraenta and Firmicutes, reducing the Bacteroidetes abundance at the phylum level, and restoring the abundances of 13 bacterial genera. In conclusion, exercise improved intestinal morphological structure damage in fluoride-exposed mice, inhibited the secretion of duodenal inflammatory factors, increased the expression of tight junctions, and alleviated the microbial disorder in mice caused by fluoride exposure for 6 months through actively regulating the composition of intestinal microorganisms and the abundance of specific bacteria.

Tunable and Contamination-Free Injection with Microfluidics by Stepinjection.

Droplet microfluidics with picoinjection provides significant advantages to multistep reactions and screenings. The T-junction design for picoinjection is convenient in adding picoliter reagents into passing droplets to initiate reactions. However, conventional picoinjectors face difficulties in eliminating cross-contamination between droplets, preventing them from widespread use in sensitive biological and molecular assays. Here, we introduce stepinjection, which uses a T-junction with a stepped channel design to elevate the diffusional buffer zone into the main channel and consequently increases the pressure difference between droplets and the inlet of the injection channel. To demonstrate the stepinjector's ability to perform contamination-sensitive enzymatic assays, we inject casein fluorescein isothiocyanate (FITC-casein) into a mixture of savinase and savinase-free (labeled with a red fluorescent dye) droplets. We observe no cross-contamination using stepinjection but find a severe cross-talk using an optimal picoinjection design. We envision that the simple, tunable, and reliable stepinjector can be easily integrated in various droplet processing devices, and facilitate various biomedical and biochemical applications including multiplex digital PCR, single-cell sequencing, and enzymatic screening.

Correction to: Description of Wenzhouxiangella salilacus sp. nov., a moderate halophilic bacterium isolated from a salt lake in Xinjiang Province, China.

In the original publication of the article, the deposit accession numbers of strain 15181T in the acknowledgment section were incorrectly provided as "KCTC 62172T and MCCC 1K03442T".

High-throughput single-cell cultivation reveals the underexplored rare biosphere in deep-sea sediments along the Southwest Indian Ridge.

Microorganisms in the deep sea play vital roles in marine ecosystems. However, despite great advances brought by high throughput sequencing and metagenomics, only a small portion of microorganisms living in the environment can be cultivated in the laboratory and systematically studied. In this study, an improved high-throughput microfluidic streak plate (MSP) platform was developed to speed up the isolation of microorganisms from deep-sea sediments and evaluated with deep-sea sediments collected from the Southwest Indian Ridge (SWIR). Based on our previously reported MSP method, we improved its isolation efficiency with a semi-automated droplet picker and improved humidity control to enable long-term cultivation with a low-nutrient medium for up to five months according to the slow-growing nature of most deep-sea species. The improved MSP method allows the isolation of microbes by selection and investigation of microbial diversity by high throughput sequencing of the pooled sample cultures. By picking individual droplets and scale-up cultivation, a total of 772 strains that were taxonomically assigned to 70 species were isolated from the deep-sea sediments in the SWIR, including 15 potential novel species. On the other hand, based on 16S rRNA gene amplicon sequencing analysis, the microbial diversity of the SWIR was studied and documented with culture-dependent and independent methods in this study. The superiority of the MSP platform in revealing the rare biosphere was also evaluated based on amplicon sequencing. The results show that droplet-based single-cell cultivation of the MSP has a much higher ability than traditional agar plate cultivation in obtaining microbial species and more than 90% of operational taxonomic units (OTUs) detected in the MSP pool belong to the rare biosphere. Our results indicate the high robustness and efficiency of the improved MSP platform in revealing the environmentally rare biosphere, especially for slow-growing species. Overall, the MSP platform has a superior ability to recover microbial diversity than conventional agar plates and it was found to hold great potential for recovering rare microbial resources from various environments.

Alkalilacustris brevis gen. nov., sp. nov., isolated from a soda lake.

A Gram-stain-negative, aerobic, non-pigmented and short-rod-shaped bacterium, designated 34079T, was isolated from a water sample of a soda lake in Jilin, a province of China. Strain 34079T grew at 10-50 °C (optimum, 35 °C), pH 7-10 (optimum, pH 8.0-8.5). NaCl was required for growth at the concentration range 1-10.0 % (w/v), with an optimum at 2.5-4 % (w/v). Chemotaxonomic analysis indicated that the sole respiratory quinone was Q-10. The predominant cellular fatty acids (>5 %) were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) and C16 : 0. The major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, three unidentified amino lipids, one unidentified amino phosphoglycolipid, one phosphoglycolipid, one unidentified glycolipid, three unidentified phospholipids and two unidentified lipids. The DNA G+C content was 65.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 34079T formed a distinct lineage in the clade of the family 'Rhodobacteraceae' with the highest sequence similarity of 96.1 % to Pararhodobacter aggregans, followed by Rhodobaca bogoriensis DSM 18756T (95.7 %) and Roseibaca ekhonensis DSM 11469T (94.7 %). The distinct biochemical, chemotaxonomic and phylogenetic differences from the previously described taxa supported that strain 34079T represents a novel species of a new genus, for which the name Alkalilacustris brevis gen. nov., sp. nov. is proposed. The type strain is 34079T (=KCTC 62428T=MCCC 1K03493T).

Description of Wenzhouxiangella salilacus sp. nov., a moderate halophilic bacterium isolated from a salt lake in Xinjiang Province, China.

A Gram-stain negative, non-motile, strictly aerobic and rod-shaped bacterium, designated as 15181T, was isolated from a salt lake in Xinjiang Province, China. Strain 15181T was able to grow at 10-40 °C (optimum 37 °C), pH 6.0-8.5 (optimum 7.0) and with 1-14% NaCl (optimum 4%, w/v). According to phylogenetic analysis based on 16S rRNA gene sequences, strain 15181T was assigned to the genus Wenzhouxiangella with high 16S rRNA gene sequence similarity of 97.4% to Wenzhouxiangella sediminis XDB06T, followed by Wenzhouxiangella marina KCTC 42284T (95.9%). Strain 15181T exhibited ANI values of 80.0% and 72.0% to W. sediminis XDB06T and W. marina KCTC 42284T, respectively. The in silico DDH analysis revealed that strain 15181T shared 19.1% and 18.7% DNA relatedness with W. sediminis XDB06T and W. marina KCTC 42284T, respectively. Chemotaxonomic analysis showed that the sole respiratory quinone was ubiquinone-8, the major fatty acids included iso-C15:0, iso-C16:0 and summed feature 9 (C16:0 10-methyl and/or iso-C17:1ω9c). The major polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified glycolipids, two unidentified phospholipids, two unidentified aminophospholipids and an unidentified lipid. On the basis of phenotypic, genotypic and chemotaxonomic characteristics presented in this study, strain 15181T is concluded to represent a novel species in the genus Wenzhouxiangella, for which the name Wenzhouxiangella salilacus sp. nov. is proposed. The type strain is 15181T (=KCTC 62172T=MCCC 1K03442T).

Rhodohalobacter barkolensis sp. nov., isolated from a saline lake and emended description of the genus Rhodohalobacter.

A Gram-stain-negative, non-motile, aerobic, rod-shaped bacterium, designated 15182T, was isolated from a saline lake in China. The novel strain 15182T was able to grow at 10-40 °C (optimum, 37 °C), pH 7.0-8.0 (optimum, 7.5) and with 0.5-4 % NaCl (optimum, 2-3 %, w/v). The phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 15182T was most closely related to the genus Rhodohalobacter by sharing the highest sequence similarity of 97.0 % with Rhodohalobacter halophilus JZ3C29T. Chemotaxonomic analysis showed that the sole respiratory quinone was menaquinone 7, the major fatty acids included C16 : 0 N alcohol and C16 : 1ω11c. The major polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four uncharacterized glycolipids, one uncharacterized phospholipid and two uncharacterized lipids. The genomic DNA G+C content of the strain 15182T was 42.4 mol%. The average nucleotide identity value between 15182T and R. halophilus JZ3C29T was 75.4 %, and the in silico DNA-DNA hybridization value of the two strains was 19.1 %. On the basis of its phenotypic, chemotaxonomic, genotypic and genomic characteristics presented in this study, strain 15182T is suggested to represent a novel species in the genus Rhodohalobacter, for which the name Rhodohalobacter barkolensis sp. nov. is proposed. The type strain is 15182T (=KCTC 62172T=MCCC 1K03442T). An emended description of the genus Rhodohalobacter is also presented.