Isolation, culture, characterization and the functional study of testicular Leydig cells from Hezuo pig.

Testosterone, an important sex hormone, regulates sexual maturation, testicular development, spermatogenesis and the maintenance of secondary sexual characteristics in males. Testicular Leydig cells are the primary source of testosterone production in the body. Hezuo pigs, native to the southern part of Gansu, China, are characterized by early sexual maturity, strong disease resistance and roughage tolerance. This study employed type IV collagenase digestion combined with cell sieve filtration to isolate and purify Leydig cells from the testicular tissue of 1-month-old Hezuo pigs. We also preliminarily investigated the functions of these cells. The results indicated that the purity of the isolated and purified Leydig cells was as high as 95%. Immunofluorescence analysis demonstrated that the isolated cells specifically expressed the 3ß-hydroxysteroid dehydrogenase antibody. Enzyme-linked immunosorbent assay results showed that the testosterone secretion of the Leydig cells cultured in vitro (generations 5-9) ranged between 1.29-1.67 ng/mL. Additionally, the content of the cellular autophagy signature protein microtubule-associated protein 1 light chain 3 was measured at 230-280 pg/mL. Through this study, we established an in vitro system for the isolation, purification and characterization of testicular Leydig cells from 1-month-old Hezuo pigs, providing a reference for exploring the molecular mechanism behind precocious puberty in Hezuo pigs.

Agreements between GLIM using left calf circumference as criterion for reduced muscle mass and PG-SGA, and GLIM using ASMI for the diagnosis of malnutrition in gastric cancer patients.

OBJECTIVE: this study aimed to explore the agreements between the Global Leadership Initiative on Malnutrition (GLIM) using left calf circumference (CC) as criterion for reduced muscle mass and the Patient-Generated Subjective Global Assessment (PG-SGA), or GLIM using appendicular skeletal muscle index (ASMI) for the diagnosis of malnutrition in gastric cancer patients. METHODS: the Nutritional Risk Screening 2002 (NRS 2002) was used as nutritional risk screening. PG-SGA and GLIM were applied for malnutrition diagnosis. Agreements were evaluated by Kappa, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, and area under the curve (AUC). RESULTS: a total of 405 gastric cancer patients were included. The values of Kappa, sensitivity, specificity, PPV, NPV, accuracy and AUC were 0.463, 67.9 %, 87.3 %, 92.9 %, 52.8 %, 73.6 % and 0.776, and 0.496, 76.7 %, 78.0 %, 89.4 %, 57.9 %, 77.0 % and 0.773, respectively, between GLIM using CC with or without NRS 2002 and PG-SGA. All values of agreement were higher than 0.800 or 80.0 % between GLIM using left CC and GLIM using ASMI. CONCLUSION: the agreements were both acceptable between GLIM using left CC and PG-SGA, and GLIM using ASMI. Left calf circumference can be one of the credible references indicating a reduced muscle mass in patients with gastric cancer.

[Retracted] Dexmedetomidine improves early postoperative neurocognitive disorder in elderly male patients undergoing thoracoscopic lobectomy.

[This retracts the article DOI: 10.3892/etm.2020.9113.].

GR/Ahi1 regulates WDR68-DYRK1A binding and mediates cognitive impairment in prenatally stressed offspring.

Accumulating research shows that prenatal exposure to maternal stress increases the risk of behavioral and mental health problems for offspring later in life. However, how prenatal stress affects offspring behavior remains unknown. Here, we found that prenatal stress (PNS) leads to reduced Ahi1, decreased synaptic plasticity and cognitive impairment in offspring. Mechanistically, Ahi1 and GR stabilize each other, inhibit GR nuclear translocation, promote Ahi1 and WDR68 binding, and inhibit DYRK1A and WDR68 binding. When Ahi1 deletion or prenatal stress leads to hyperactivity of the HPA axis, it promotes the release of GC, leading to GR nuclear translocation and Ahi1 degradation, which further inhibits the binding of Ahi1 and WDR68, and promotes the binding of DYRK1A and WDR68, leading to elevated DYRK1A, reduced synaptic plasticity, and cognitive impairment. Interestingly, we identified RU486, an antagonist of GR, which increased Ahi1/GR levels and improved cognitive impairment and synaptic plasticity in PNS offspring. Our study contributes to understanding the signaling mechanisms of prenatal stress-mediated cognitive impairment in offspring.

Regulatory Effects of the Kiss1 Gene in the Testis on Puberty and Reproduction in Hezuo and Landrance Boars.

Kisspeptin, a neuropeptide encoded by the Kiss1 gene, combines with its receptor Kiss1R to regulate the onset of puberty and male fertility by the hypothalamic-pituitary-gonadal axis. However, little is known regarding the expression signatures and molecular functions of Kiss1 in the testis. H&E staining revealed that well-arranged spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa, were observed in 4-, 6-, and 8-month-old testes compared to 1- and 3-month-old testes of Hezuo pigs; however, these were not observed in Landrance until 6 months. The diameter, perimeter, and cross-sectional area of seminiferous tubules and the perimeter and area of the tubular lumen increased gradually with age in both pigs. Still, Hezuo pigs grew faster than Landrance. The cloning results suggested that the Hezuo pigs' Kiss1 CDS region is 417 bp in length, encodes 138 amino acids, and is highly conserved in the kisspeptin-10 region. qRT-PCR and Western blot indicated that the expression trends of Kiss1 mRNA and protein were essentially identical, with higher expression levels at post-pubertal stages. Immunohistochemistry demonstrated that the Kiss1 protein was mainly located in Leydig cells and post-pubertal spermatogenic cells, ranging from round spermatids to spermatozoa. These studies suggest that Kiss1 is an essential regulator in the onset of puberty and spermatogenesis of boars.

Molecular characterization, expression patterns and cellular localization of BCAS2 gene in male Hezuo pig.

Background: Breast carcinoma amplified sequence 2 (BCAS2) participates in pre-mRNA splicing and DNA damage response, which is implicated in spermatogenesis and meiosis initiation in mouse. Nevertheless, the physiological roles of BCAS2 in the testes of large mammals especially boars remain largely unknown. Methods: In this study, testes were collected from Hezuo pig at three development stages including 30 days old (30 d), 120 days old (120 d), and 240 days old (240 d). BCAS2 CDS region was firstly cloned using RT-PCR method, and its molecular characteristics were identified using relevant bioinformatics software. Additionally, the expression patterns and cellular localization of BCAS2 were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry and immunofluorescence. Results: The cloning and sequence analysis indicated that the Hezuo pig BCAS2 CDS fragment encompassed 678 bp open reading frame (ORF) capable of encoding 225 amino acid residues, and possessed high identities with some other mammals. The results of qRT-PCR and Western blot displayed that BCAS2 levels both mRNA and protein were age-dependent increased (p < 0.01). Additionally, immunohistochemistry and immunofluorescence results revealed that BCAS2 protein was mainly observed in nucleus of gonocytes at 30 d testes as well as nucleus of spermatogonia and Sertoli cells at 120 and 240 d testes. Accordingly, we conclude that BCAS2 is critical for testicular development and spermatogenesis of Hezuo pig, perhaps by regulating proliferation or differentiation of gonocytes, pre-mRNA splicing of spermatogonia and functional maintenance of Sertoli cells, but specific mechanism still requires be further investigated.

Cloning, expression analysis and localization of DAZL gene implicated in germ cell development of male Hezuo pig.

Deleted in azoospermia-like (DAZL) is essential for mammalian testicular function and spermatogenesis. To explore the molecular characterization, expression patterns, and cellular localization of the DAZL in Hezuo pig testes, testicular tissue was isolated from Hezuo pig at five development stages including 30 days old (30 d), 90 days old (90 d), 120 days old (120 d), 180 days old (180 d), and 240 days old (240 d). DAZL cDNA was first cloned using the RT-PCR method, and its molecular characterization was analyzed using relevant bioinformatics software. Subsequently, the expression patterns and cellular localization of DAZL were evaluated using quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The cloning and sequence analysis showed that the Hezuo pig DAZL cDNA fragment contained 888 bp open reading frame (ORF) capable of encoding 295 amino acid residues and exhibited high identities with some other mammals. The qRT-PCR and Western blot results indicated that DAZL was specifically expressed in Hezuo pig testes, and DAZL levels of both mRNA and protein were expressed at all five reproductive stages of Hezuo pig testes, with extremely significant higher expression levels in 90 d, 120 d, 180 d, and 240 d than those in 30 d (p < 0.01). Additionally, immunohistochemistry results revealed that DAZL protein was mainly localized in gonocytes at 30 d testes, primary spermatocytes, and spermatozoon at other developmental stages, and Leydig cells throughout five development stages. Together, these results suggested that DAZL may play an important role by regulating the proliferation or differentiation of gonocytes, development of primary spermatocytes and spermatozoon, and functional maintenance of Leydig cells in testicular development and spermatogenesis of Hezuo pig. Nevertheless, the specific regulatory mechanisms underlying these phenomena still requires further investigated and verified.

TNF-α and IL-8 levels are positively correlated with hypobaric hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats.

The expression status of proinflammatory cytokines in high-altitude pulmonary arterial hypertension (PAH) has been well studied. However, the changes in interleukin (IL)-8 and tumor necrosis factor α (TNF-α) during the reversible changes in pulmonary vascular remodeling (PVR) in PAH after detaching from a hypobaric hypoxic environment have not been elucidated. This investigation elucidated a high-altitude PAH rat model. Then, PAH rats in the high-altitude group were maintained in the high-altitude area, and rats in the low-altitude group returned to the low-altitude area. After 0, 10, 20, and 30 days of PAH modeling, right ventricular systolic pressure (RVSP) and the mean pulmonary arterial pressure (mPAP) were assessed. Right ventricular (RV) hypertrophy was reflected by the ratio of RV/[left ventricle + interventricular septum (S)]. Pathological changes in PVR were accessed by hematoxylin-eosin staining, and medial wall thickness (WT%) and medial wall area (WA%) were measured. TNF-α and IL-8 levels in pulmonary artery tissues and blood were measured with Western blot assay and enzyme-linked immunosorbent assay, respectively. Our results showed that PAH rats exhibited a substantial increase in RVSP and mPAP, RV hypertrophy, PVR, and enhanced generation of TNF-α and IL-8. Then, we found that these pathological changes were gradually aggravated and TNF-α and IL-8 levels were increased in rats in the high-altitude group after 10, 20, and 30 days of PAH modeling. In contrast, the mPAP was decreased and PVR was alleviated in rats in the low-altitude group, accompanying with reduced TNF-α and IL-8 production. In conclusion, our study demonstrated that the generation of TNF-α and IL-8 was also reversible during the reversible changes in PVR after detaching from a hypobaric hypoxic environment. Thus, proinflammatory cytokine TNF-α and IL-8 levels are positively correlated with PVR severity.

Short-term outcomes of an enhanced recovery after surgery pathway for children with congenital scoliosis undergoing posterior spinal fusion: a case-control study of 70 patients.

Increasing evidence demonstrates the advantages of an enhanced recovery after surgery (ERAS) protocol; however, few studies have evaluated ERAS in pediatric patients. This study aimed to evaluate the effect of ERAS in pediatric patients with congenital scoliosis. Seventy pediatric patients with congenital scoliosis underwent posterior hemivertebra resection and fusion with pedicle screws and were prospectively randomly assigned to the ERAS group (n = 35) and control group (n = 35). ERAS management comprised 15 elements including a shortened fasting time, optimized anesthesia protocol, and multimodal analgesia. The control group received traditional perioperative management. Clinical outcome was evaluated by hospital stay, surgery-related indicators, diet, pain scores, laboratory tests, and complications. The surgical outcome showed a similar correction rate in the ERAS group (84.0%) and control group (89.0%; P = 0.471). The mean fasting time was significantly shorter in the ERAS group than in the control group. Compared with the control group, the ERAS group had significantly shorter mean times to postoperative hospital stay, first anal exhaust and defecation, significantly lower mean pain scores in the first 2 days postoperatively (P < 0.05), and a significantly lower mean interleukin-6 concentration on postoperative day 1 (P < 0.001). The incidence of complications was similar in the ERAS group and control group (P > 0.05). The ERAS protocol is effective and safe for pediatric patients with congenital spinal deformity and may significantly improve the treatment efficacy compared with traditional perioperative management methods. Levels of Evidence: III.

Optimized Traffic Light System with AIC and Application to the 2021 M6.7 Yangbi Earthquake Sequence.

One important question in earthquake prediction is whether a moderate or large earthquake will be followed by an even bigger one. Through temporal b-value evolution analysis, the traffic light system can be used to estimate if an earthquake is a foreshock. However, the traffic light system does not take into account the uncertainty of b-values when they constitute a criterion. In this study, we propose an optimization of the traffic light system with the Akaike Information Criterion (AIC) and bootstrap. The traffic light signals are controlled by the significance level of the difference in b-value between the sample and the background rather than an arbitrary constant. We applied the optimized traffic light system to the 2021 Yangbi earthquake sequence, which could be explicitly recognized as foreshock-mainshock-aftershock using the temporal and spatial variations in b-values. In addition, we used a new statistical parameter related to the distance between earthquakes to track earthquake nucleation features. We also confirmed that the optimized traffic light system works on a high-resolution catalog that includes small-magnitude earthquakes. The comprehensive consideration of b-value, significance probability, and seismic clustering might improve the reliability of earthquake risk judgment.

Photocatalytic Hydrogen Production Activity and Mechanism of New Nickel-Based Sulfur Complexes in Aqueous Solution.

The development of industry and the increase in population have caused energy shortages and environmental pollution problems. Developing clean and storable new energy is identified as a key way to solve the problems above. Hydrogen is viewed as the most potential energy carrier due to its high calorific value and pollution-free. To convert solar energy into hydrogen energy, three nickel-based catalysts, Ni(aps)(pys)2 (aps=2-amino-2-phenylacetic salicylaldehyde) (1), Ni(ads)(pys)2 (ads=aniline salicylaldehyde, pys=pyridine-2-thiolate) (2), Ni(acs)(pys)2 (acs=aniline 5-chlorosalicylaldehyde) (3), were synthesized and explored as photocatalysts for hydrogen production. A three-component photocatalytic system for hydrogen production was constructed using target complex as photocatalyst, triethanolamine (TEOA) as electron sacrificial agent and fluorescein (FL) as photosensitizer. Under the optimum conditions, about 1504 µmol of H2 can be obtained with 25 mg catalyst 2 after 3 hours of irradiation. Finally, the hydrogen-production mechanism was discussed by experimental and theoretical methods.

The mitochondrial Ahi1/GR participates the regulation on mtDNA copy numbers and brain ATP levels and modulates depressive behaviors in mice.

BACKGROUND: Previous studies have shown that depression is often accompanied by an increase in mtDNA copy number and a decrease in ATP levels; however, the exact regulatory mechanisms remain unclear. METHODS: In the present study, Western blot, cell knockdown, immunofluorescence, immunoprecipitation and ChIP-qPCR assays were used to detect changes in the Ahi1/GR-TFAM-mtDNA pathway in the brains of neuronal Abelson helper integration site-1 (Ahi1) KO mice and dexamethasone (Dex)-induced mice to elucidate the pathogenesis of depression. In addition, a rescue experiment was performed to determine the effects of regular exercise on the Ahi1/GR-TFAM-mtDNA-ATP pathway and depression-like behavior in Dex-induced mice and Ahi1 KO mice under stress. RESULTS: In this study, we found that ATP levels decreased and mitochondrial DNA (mtDNA) copy numbers increased in depression-related brain regions in Dex-induced depressive mice and Ahi1 knockout (KO) mice. In addition, Ahi1 and glucocorticoid receptor (GR), two important proteins related to stress and depressive behaviors, were significantly decreased in the mitochondria under stress. Intriguingly, GR can bind to the D-loop control region of mitochondria and regulate mitochondrial replication and transcription. Importantly, regular exercise significantly increased mitochondrial Ahi1/GR levels and ATP levels and thus improved depression-like behaviors in Dex-induced depressive mice but not in Ahi1 KO mice under stress. CONCLUSIONS: In summary, our findings demonstrated that the mitochondrial Ahi1/GR complex and TFAM coordinately regulate mtDNA copy numbers and brain ATP levels by binding to the D-loop region of mtDNA Regular exercise increases the levels of the mitochondrial Ahi1/GR complex and improves depressive behaviors. Video Abstract.

Thrombin Determination Using Graphene Oxide Sensors with Co-Assisted Amplification.

Graphene oxide (GO) is widely used in sensors. The detection of proteins based on bare GO has been developed; however, the detection sensitivity needs to be improved. In this paper, a novel GO-DNA sensor for thrombin detection was developed using an aptamer linked to the surface of GO. Polyethylene glycol (PEG) was further used to prevent thrombin from nonspecific adsorption and to improve the sensitivity of the sensor for detection of thrombin. In order to improve the limit of detection for thrombin, we developed a GO and RecJf exonuclease co-assisted signal amplification strategy, and a detection limit of 24.35 fM for thrombin was achieved using this strategy. The results show that it is a promising method in analytical applications.

Ahi1 regulates serotonin production by the GR/ERß/TPH2 pathway involving sexual differences in depressive behaviors.

BACKGROUND: Depression is one of the most common psychiatric diseases. The monoamine transmitter theory suggests that neurotransmitters are involved in the mechanism of depression; however, the regulation on serotonin production is still unclear. We previously showed that Ahi1 knockout (KO) mice exhibited depression-like behavior accompanied by a significant decrease in brain serotonin. METHODS: In the present study, western blot, gene knockdown, immunofluorescence, dual-luciferase reporter assay, and rescue assay were used to detect changes in the Ahi1/GR/ERß/TPH2 pathway in the brains of male stressed mice and male Ahi1 KO mice to explain the pathogenesis of depression-like behaviors. In addition, E2 levels in the blood and brain of male and female mice were measured to investigate the effect on the ERß/TPH2 pathway and to reveal the mechanisms for the phenomenon of gender differences in depression-like behaviors. RESULTS: We found that the serotonin-producing pathway-the ERß/TPH2 pathway was inhibited in male stressed mice and male Ahi1 KO mice. We further demonstrated that glucocorticoid receptor (GR) as a transcription factor bound to the promoter of ERß that contains glucocorticoid response elements and inhibited the transcription of ERß. Our recent study had indicated that Ahi1 regulates the nuclear translocation of GR upon stress, thus proposing the Ahi1/GR/ERß/TPH2 pathway for serotonin production. Interestingly, female Ahi1 KO mice did not exhibit depressive behaviors, indicating sexual differences in depressive behaviors compared with male mice. Furthermore, we found that serum 17ß-estradiol (E2) level was not changed in male and female mice; however, brain E2 level significantly decreased in male but not female Ahi1 KO mice. Further, ERß agonist LY-500307 increased TPH2 expression and 5-HT production. Therefore, both Ahi1 and E2 regulate the ERß/TPH2 pathway and involve sexual differences in brain serotonin production and depressive behaviors. CONCLUSIONS: In conclusion, although it is unclear how Ahi1 controls E2 secretion in the brain, our findings demonstrate that Ahi1 regulates serotonin production by the GR/ERß/TPH2 pathway in the brain and possibly involves the regulation on sex differences in depressive behaviors. Video Abstract.

Temporal Variation of b Value with Statistical Test in Wenchuan Area, China Prior to the 2008 Wenchuan Earthquake.

The Gutenberg-Richter b value describes the ratio between large and small events. A number of studies have suggested that the b value decreases before large earthquakes. In this study, we investigate the temporal variation of the b value of an area along the main rupture zone of the 2008 Wenchuan earthquake (M8.0) prior to the great event. Before estimating b values, we tested the earthquake catalog to make sure that we use the reliable frequency-magnitude distribution by the calculation of MC (completeness of magnitude). We define parameter P (ΔAIC ≧ 2) values to examine the significance level of b-value changes in the temporal variation by combining a boostrap method with Akaike's Information Criterion (AIC). The b value in the main rupture zone shows a long-term decrease trend. We then focus on a smaller area where the initial rupture starts. The results show that b values significantly changed about 3 months before the 2008 Wenchuan earthquake in the initial rupture area, indicating that the b value has a potential capability to monitor and detect precursory phenomena of great earthquakes.

Short-Term Instantaneous Prophylaxis and Efficient Treatment Against SARS-CoV-2 in hACE2 Mice Conferred by an Intranasal Nanobody (Nb22).

Current COVID-19 vaccines need to take at least one month to complete inoculation and then become effective. Around 51% of the global population is still not fully vaccinated. Instantaneous protection is an unmet need among those who are not fully vaccinated. In addition, breakthrough infections caused by SARS-CoV-2 are widely reported. All these highlight the unmet needing for short-term instantaneous prophylaxis (STIP) in the communities where SARS-CoV-2 is circulating. Previously, we reported nanobodies isolated from an alpaca immunized with the spike protein, exhibiting ultrahigh potency against SARS-CoV-2 and its variants. Herein, we found that Nb22, among our previously reported nanobodies, exhibited ultrapotent neutralization against Delta variant with an IC50 value of 0.41 ng/ml (5.13 pM). Furthermore, the crystal structural analysis revealed that the binding of Nb22 to WH01 and Delta RBDs both effectively blocked the binding of RBD to hACE2. Additionally, intranasal Nb22 exhibited protection against SARS-CoV-2 Delta variant in the post-exposure prophylaxis (PEP) and pre-exposure prophylaxis (PrEP). Of note, intranasal Nb22 also demonstrated high efficacy against SARS-CoV-2 Delta variant in STIP for seven days administered by single dose and exhibited long-lasting retention in the respiratory system for at least one month administered by four doses, providing a strategy of instantaneous short-term prophylaxis against SARS-CoV-2. Thus, ultrahigh potency, long-lasting retention in the respiratory system and stability at room-temperature make the intranasal or inhaled Nb22 to be a potential therapeutic or STIP agent against SARS-CoV-2.

Accelerating Nickel-Based Molecular Construction via DFT Guidance for Advanced Photocatalytic Hydrogen Production.

Understanding the nickel-based molecular catalyst structure and functional relationship is crucial for catalytic hydrogen production in aqueous solutions. Density functional theory (DFT) provides mature theoretical knowledge for efficient catalyst design, significantly reducing catalyst synthesis time and energy consumption. In the present work, three molecular catalysts, Ni(qbz)(pys)2 (qbz = 2-quinoline benzimidazole) (NQP 1), Ni(qbo)(pys)2 (qbo = 2-quinoline benzothiazole) (NQP 2), and Ni(pbz)(pys)2 (pbz = 4-chloro-2,2-pyridylbenzimidazole) (NQP 3) (pys = 2-mercaptopyridine), were designed and synthesized and exhibit a high performance for H2 generation in aqueous solution with a lamp (λ ≥ 400 nm) under visible light irradiation. Under the optimal conditions, a H2 evolution rate as high as 1190 µmol h-1 can be obtained over 25 mg of NQP 1 with the best catalytic performance. DFT has been adopted in this study to unveil the relationship between the ligand qbz and catalyst NQP 1─an efficient step in the design of catalysts with an excellent catalytic performance. We show that, in addition to the presence of the triphenyl ring increasing the overall electron density, rapid electron transfer (ET) from excited fluorescein (Fl) to NQP 1 significantly improves the chance of photogenerated electrons transferring to the active site, ultimately increasing the catalytic activity for H2 production. This work on understanding the correlation between structures and properties of complexes provides a new idea for manufacturing high-performance photocatalysts.

Neck and mediastinal hematoma caused by a foreign body in the esophagus with diagnostic difficulties: A case report.

BACKGROUND: Esophageal foreign body (FB) is a common clinical emergency. Clinically, computed tomography (CT) scans are important in the diagnosis of FBs in the esophagus. Here, we report a case of esophageal perforation and cervical hematoma, caused by a FB, whose uniqueness made rapid diagnosis difficult. CASE SUMMARY: A 42-year-old man was transferred to our hospital with esophageal perforation, which was accompanied by cervical and mediastinal hematoma. CT scans only revealed a black shadow, approximately 2.5 cm in diameter, in the upper esophagus. After multidisciplinary discussion, he was quickly subjected to mediastinal hematoma resection, peripheral nerve compression release, esophageal FB removal and esophagectomy. Eventually, we removed a small crab with a pointed tip from his esophagus. CONCLUSION: This was an unusual case of occurrence of sharp polygonal esophageal FBs caused by a small crab. Rapid diagnosis of this FB was difficult, mainly due to its translucent nature. Occurrence of sharp FBs, with cavities that sometimes only appear as black shadows on CT scans, can easily be mistaken for esophageal lumens. More attention should be paid to such sharp polygonal FBs.

Antibiotic resistance genes in gut of breast-fed neonates born by caesarean section originate from breast milk and hospital ward air.

The human gut is a reservoir of antibiotic resistance genes (ARGs). Even in the absence of antibiotics, ARGs are present in large quantities in faeces of adults, children and even newborns. However, where and when ARGs are acquired remains unclear, as does the types of ARGs acquired. Herein, we recruited 82 pairs of women and their caesarean section newborns. Conventional culture methods and quantitative PCR were employed to detect nine species and six ARG types in meconia, faeces from 3-day-old newborns, amniotic fluid, colostrum, and hospital ward air samples. Furthermore, ARG transfer was explored by tracking Staphylococcus epidermidis isolated from faeces of 3-day-old newborns, colostrum and ward air samples using multi-locus sequence typing (MLST). No ARGs or microorganisms were detected in meconia or amniotic fluid. One or more ARGs were detected in 90.2% of faeces from 3-day-old newborns, and the mecA gene exhibited the highest detection rate (45.1%). ARGs were detected in 85.4% of colostra consistent with ARGs in faeces from 3-day-old newborns. Some ARGs were detected in ward air, and might also be a source of ARGs in neonatal faeces. Isolation of S. epidermidis from neonatal faeces was consistent with antibiotic resistance and gene profiles for colostrum samples. Traceability analysis of S. epidermidis showed that ARGs in neonatal faeces mainly originated from colostrum, and partly from ward air. After birth, neonates born by caesarean section obtain a variety of ARGs mainly from colostrum, and partly from ward air.

Effects of keratinocyte-derived and fibroblast-derived exosomes on human epidermal melanocytes.

BACKGROUND: Exosomes have been demonstrated to carry proteins, membrane lipids, mRNAs and microRNAs which can be transferred to surrounding cells and regulate the functions of those recipient cells. OBJECTIVES: The objective of the study was to investigate the effects of exosomes released by keratinocytes and fibroblasts on the proliferation, tyrosinase activity and melanogenesis of melanocytes. METHODS: Melanocytes, keratinocytes and fibroblasts obtained from human foreskin were cultured and exosomes secreted by keratinocytes and fibroblasts were harvested from the culture supernatants by ultracentrifugation. Each exosome fraction was divided into two parts; one part was subjected to high-throughput sequencing using an Illumina HiSeq sequencer to characterize the microRNA expression profiles, while the other part was labeled with the fluorescent dye PKH67 and was then co-cultivated with epidermal melanocytes. RESULTS: High-throughput sequencing analysis showed 168 differentially expressed microRNA within exosomes derived from keratinocytes and from fibroblasts, 97 of those being up-regulated with the other 71 down-regulated. Gene ontology analysis showed that the target genes responsible for these differentially expressed microRNAs were mainly enriched in the protein-binding region of molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that target genes regulated by differentially expressed microRNA were mainly involved in mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, cAMP signaling pathway and Wnt signaling pathway. Keratinocyte-derived exosomes were taken up by melanocytes co-cultured with them and promoted the proliferation, tyrosinase activity and melanin synthesis of those melanocytes. However, fibroblast-derived exosomes had no similar effects on melanocytes. CONCLUSION: Keratinocyte-derived exosomes but not fibroblast-derived exosomes were taken up by melanocytes in co-culture and significantly stimulated their proliferation, tyrosinase activity and melanin synthesis. Those different effects may be mainly due to the differential expression of microRNAs in exosomes derived from the different types of cells. LIMITATIONS: Electron microscopy of the obtained exosomes and in-depth study of apparently differentially expressed microRNAs were not performed.