METTL3/N6‐methyladenosine/ miR‐21‐5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF‐κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR‐21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF‐kB signalling pathway. However, whether miR‐21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6‐methyladenosine (m⁶A) modification‐dependent primary microRNA (pri‐microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR‐21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK‐2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR‐21‐5p mimic or miR‐21‐5p inhibitor. We found that the levels of miR‐21‐5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF‐kB pathway was activated by miR‐21‐5p, confirming that miR‐21‐5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR‐21‐5p maturation. Our research is the first to demonstrate the role of the METTL3‐m⁶A‐miR‐21‐5p‐SPRY1/ERK/NF‐kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

Otoacoustic emissions,auditory evoked potentials and self-reported gender in people affected by disorders of sex development (DSD)

Both otoacoustic emissions (OAEs) and auditory evoked potentials (AEPs) are sexually dimorphic, and both are believed to be influenced by prenatal androgen exposure. OAEs and AEPs were collected from people affected by 1 of 3 categories of disorders of sex development (DSD) — (1) women with complete androgen insensitivity syndrome (CAIS); (2) women with congenital adrenal hyperplasia (CAH); and (3) individuals with 46,XY DSD including prenatal androgen exposure who developed a male gender despite initial rearing as females (men with DSD). Gender identity (GI) and role (GR) were measured both retrospectively and at the time of study participation, using standardized questionnaires. The main objective of this study was to determine if patterns of OAEs and AEPs correlate with gender in people affected by DSD and in controls. A second objective was to assess if OAE and AEP patterns differed according to degrees of prenatal androgen exposure across groups. Control males, men with DSD, and women with CAH produced fewer spontaneous OAEs (SOAEs) – the male-typical pattern – than control females and women with CAIS. Additionally, the number of SOAEs produced correlated with gender development across all groups tested. Although some sex differences in AEPs were observed between control males and females, AEP measures did not correlate with gender development, nor did they vary according to degrees of prenatal androgen exposure, among people with DSD. Thus, OAEs, but not AEPs, may prove useful as bioassays for assessing early brain exposure to androgens and predicting gender development in people with DSD.     

中缅树鼩消化道长度和重量变化

为探讨栖息于昆明禄劝地区中缅树鼩(Tupaia belangeri)的消化道特征与环境之间的适应关系,在2008年6月和12月(夏季和冬季)分别对自然环境中缅树鼩的胃、小肠、大肠、盲肠的长度、含内容物重、去内容物重、干组织重等消化道指标进行了测定。结果表明,中缅树鼩消化道特征冬季和夏季存在变化,随着温度降低、食物质量下降,中缅树鼩的小肠长度和重量增加;各器官重量均在冬季最大;中缅树鼩在受到低温、食物质量下降等因子胁迫下,通过调节消化道长度和重量来满足能量需求的增加,维持正常的生理机能。中缅树鼩的消化道在冬季和夏季中表现出的变化模式及消化对策对其在自然环境中的生存是至关重要的。     

Photonic characteristics and ex vivo imaging of Escherichia coli-Xen14 within the bovine reproductive tract

The objectives of this study were to (1) characterize the photonic properties of Escherichia coli-Xen14 and (2) conduct photonic imaging of E. coli-Xen14 within bovine reproductive tract segments (RTS) ex vivo (Bos indicus). E. coli-Xen14 was grown for 24 h in Luria Bertani medium (LB), with or without kanamycin (KAN). Every 24 h, for an 8-d interval, inoculums were imaged and photonic emissions (PE) collected. Inoculums were subcultured and plated daily to determine the colony forming units (CFU) and ratio of photon emitters to nonemitters. In the second objective, abattoir-derived bovine reproductive tracts (n = 9) were separated into posterior and anterior vagina, cervix, uterine body, and uterine horns. Two concentrations (3.2 × 10⁸ and 3.2 × 10⁶ CFU/200 μL for relative [High] and [Low], respectively) of E. coli-Xen14 were placed in translucent tubes for detection of PE through RTS. The CFU did not differ (P = 0.31) over time with or without KAN presence; they remained stable with 99.93% and 99.98% photon emitters, respectively. However, PE were lower (P < 0.0001) in cultures containing KAN than in those containing no KAN (629.8 ± 117.7 vs. 3012.0 ± 423.5 relative lights units per second [RLU/sec], respectively). On average, the percentage of PE between RTS, for both concentrations, was higher (P < 0.05) in the uterine body. In summary, E. coli-Xen14 remained stable with respect to the proportions of photon emitters with or without KAN (used to selectively culture E. coli-Xen14). However, KAN presence suppressed photonic activity. The ability to detect PE through various segments of the reproductive tract demonstrated the feasibility of monitoring the presence of E. coli-Xen14 in the bovine reproductive tract ex vivo.     

A multiplex PCR method to detect 14 Escherichia coli serogroups associated with urinary tract infections

Urinary tract infections (UTIs) are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC). E. coli strains belonging to 14 serogroups, including O1, O2, O4, O6, O7, O8, O15, O16, O18, O21, O22, O25, O75 and O83, are the most frequently detected UPEC strains in a diverse range of clinical urine specimens. In the current study, the O-antigen gene clusters of E. coli serogroups O1, O2, O18 and O75 were characterized. A multiplex PCR method based on O-antigen-specific genes was developed for the simultaneous detection of all 14 E. coli serogroups. The multiplex PCR method was shown to be highly specific and reproducible when tested against 186 E. coli and Shigella O-serogroup reference strains, 47 E. coli clinical isolates and 10 strains of other bacterial species. The sensitivity of the multiplex PCR method was analyzed and shown to detect O-antigen-specific genes in samples containing 25 ng of genomic DNA or in mock urine specimens containing 40 colony-forming units (CFUs) per ml. Five urine specimens from hospital were examined using this multiplex PCR method, and the result for one sample was verified by the conventional serotyping methods. The multiplex PCR method developed herein can be used for the detection of relevant E. coli strains from clinical and/or environmental samples, and it is particularly useful for epidemiologic analysis of urine specimens from patients with UTIs.     

Enhancement of the innate immune response of bladder epithelial cells by Astragalus polysaccharides through upregulation of TLR4 expression

The innate host defenses at mucosal surfaces are critical in the early stages of urinary tract bacterial infection. Recent studies have shown that uroepithelial cells aid innate immune cells in fighting off infection, although the exact mechanism by which the uroepithilium participates in immunity remains unclear. TLR4 has been implicated to possess antimicrobial activities specific for bladder epithelial cells (BECs). TLR4 promotes secretion of IL-6 and IL-8, mediates inhibition of bladder epithelial cell (BEC) bacterial invasion, and mediates expulsion of uropathogenic Escherichia coli from BECs. In this study, cultured 5637 cells and Balb/C mice were treated with Astragalus polysaccharides (APS) against invading E. coli. To determine the contribution of TLR4 upregulation to immune response, TLR4 expression and bacterial colony numbers were monitored. After 24 h incubation, only 5637 cells treated with 500 μg/ml APS expressed higher levels of TLR4 compared with the untreated group. However, after 48 h, all 5637 cells treated by APS showed higher levels of TLR4 expression than the control cells. The TLR4 expression in the bladder and macrophages mice that received APS was higher than that in the controls. Bacterial colonization in 5637 cells and the bladders of mice treated with APS was significantly reduced compared with the controls. These results demonstrate that at certain concentrations, APS can induce increased TLR4 expression in vivo and in vitro. Further, TLR4 expression upregulation can enhance innate immunity during mucosal bacterial infection. The findings establish the use of APS to modulate the innate immune response of the urinary tract through TLR4 expression regulation as an alternative option for UTI treatment.     

Mutations in VEGFA are associated with congenital left ventricular outflow tract obstruction

Left ventricular outflow tract obstruction (LVOTO) comprises a spectrum of stenotic lesions. Previous studies have shown that the vascular endothelial growth factor (VEGF) signaling system plays a critical role in cardiac cushion formation, vasculogenesis, and angiogenesis. We hypothesize that VEGFA may be a potential candidate gene associated with the spectrum of LVOTO lesions. However, it remains unclear whether the VEGFA gene is responsible for the development of LVOTO malformations. In this study, we identified three exon mutations in the VEGFA gene in three of 192 nonsyndromic LVOTO patients, and the overall mutation frequency was 1.6% (3/192). The c.454C>T (p.Arg152X) nonsense mutation and c.19_22dupGACA (p.Thr8ArgfsX78) internal tandem duplication mutation each introduced a premature stop codon and are predicted to produce a truncated VEGFA protein. The c.998G>A missense mutation changes a highly conserved arginine to a glutamine at residue 333 (p.Arg333Gln). These mutations were carried by some family members, and average penetrance was 33.3%. The present study suggests, for the first time to our knowledge, that VEGFA mutations may be associated with congenital LVOTO malformations. We provide evidence that LVOTO is likely oligogenic.     

The Pollen Tube Pathway in Tasmannia insipida (Winteraceae): Homology of the Male Gametophyte Conduction Tissue in Angiosperms

Abstract: The pathway of the pollen tube in Tasmannia insipida (Winteraceae), an archaic angiosperm, is described. Visual observations and measurements of percentage sugar content from stigmatic exudate and the calyptra droplet were made. The droplet that forms on the calyptra of unopened flowers was artificially pollinated, but no pollen tubes appeared to grow through it to the gynoecium of female flowers. Calyptra droplets occur on both female and male (having reduced female structures) flowers of this dioecious plant. It is considered unlikely that a drop pollination mechanism, analogous to that found in some gymnosperms, is at work in T. insipida, and it is proposed that one possible droplet function is as a "reward" to potential pollinators in advance of flower opening. Pollen tubes were observed to grow almost exclusively along epidermal cells. Early thoughts on angiosperm transmission tissue are re-examined and built upon. It is proposed that the male gametophyte conduction tissue in angiosperms is homologous with epidermal tissue, and that pollen tube passage occurred originally solely along the surface of specialized epidermal cells, or possibly for short distances along unspecialized regions covered with exudate derived from these cells. Some earlier attempts to explain transmitting tissue homology and evolution are discussed.     

Effects of diet quality on phenotypic flexibility of organ size and digestive function in Mongolian gerbils (Meriones unguiculatus)

In the context of evolution and ecology, there is a trade-off between the benefits of processing food through a digestive system with specific phenotypic attributes and the cost of maintaining and carrying the digestive system. In this study, we tested the hypothesis that digestive modulations at several levels can match each other to meet the energy and nutrient demands of Mongolian gerbils, a small granivorous rodent species, by acclimating them to a high-quality diet diluted with alfalfa powder. Mongolian gerbils on the diluted diet maintained metabolizable energy intake by an integrated processing response (IPR), which included increases in dry matter intake, gut capacity and rate of digesta passage after 2-weeks of acclimation. Down-regulation of hydrolytic enzyme activity in the intestinal brush-border membrane supported the adaptive modulation hypothesis. The absence of up-modulation of summed enzyme hydrolytic capacity on the diluted diet indicated that greater mass of small intestine on a high-fibre diet is not a direct indicator of digestive or absorptive capacity. Changes in mass of vital organs and carcass suggested that the amount of energy allocated to various organs and hence physiological functions was regulated in response to diet shift.     

Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that k(on)-driven mutations are more likely to initiate non-specific binding events than k(off)-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.