COVID-19 vs. Influenza: A Chest X-ray Comparison.

Introduction COVID-19 and influenza are primarily respiratory diseases, have similar symptoms with most patients developing mild to moderate illness, and show similar features on chest X-rays. We hypothesize that patients seeking treatments at the emergency department (ED) due to COVID-19 or influenza infection will have similar severity levels of features on chest X-rays, with most of them demonstrating normal to mildly abnormal chest X-ray findings.  Methods Chest X-ray images of 312 COVID-19 patients and 312 influenza patients were obtained from the teaching files of a general diagnostic radiologist. Images from each of these two groups were reviewed and classified. Based on the severity levels of lung abnormalities, each image was categorized into one of four categories: normal, mildly abnormal, moderately abnormal, or severely abnormal. The total number of images in each category within each disease group was counted, and the percentage was calculated compared to the total number of images analyzed in that group. Results from both groups were then compared. Results The severity levels of chest X-ray abnormalities were similar between the COVID-19 group and the COVID-negative influenza group at the time of ED visits, with most images being normal or mildly abnormal. The percentages of the images categorized as normal, mildly abnormal, moderately abnormal, and severely abnormal in the COVID-19 group and the influenza group were 38-39%, 28-29%, 22-21%, and 12-11%, respectively. Conclusion Our findings suggest that in the ED setting, no distinction can be made between COVID-19 and Influenza infections if based just on chest X-rays.

Retinoic Acid Regulates Calcium Signaling to Promote Mouse Ovarian Granulosa Cell Proliferation.

Normal development of ovarian follicles is critical for female reproduction and endocrine function. We have identified retinoic acid (RA) and the RA-degrading enzyme CYP26B1 as regulators of ovarian follicle development and showed that RA and a CYP26 inhibitor stimulated ovarian granulosa cell proliferation. The mechanism underpinning RA-dependent proliferation, however, is not known. The current study was designed to examine the role of intracellular calcium (Ca2+) signaling in mediating the effects of RA on primary mouse granulosa cell proliferation. In single-cell Ca2+ imaging experiments, treatment of cultured granulosa cells with RA increased the steady-state Ca2+ content of the endoplasmic reticulum (ER) stores. This correlated with increased store-operated Ca2+ entry (SOCE) and enhanced inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca2+ release. In proliferation assays, RA treatment or Cyp26b1 knockdown stimulated proliferation, whereas Cyp26b1 overexpression inhibited proliferation. When RA was given together with 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3R-dependent ER Ca2+ release and SOCE, with xestospongin C, a selective IP3R- receptor antagonist, or with 3,5-bis (trifluoromethyl)pyrazole (BTP-2), a specific SOCE blocker, the stimulatory effect of RA on cell proliferation was abolished. Further investigation showed that treatment with 2-APB or BTP-2 inhibited RA induction of RA response element (RARE) activation in granulosa cells, confirming an important role for Ca2+ signaling in mediating RA actions. Overall, these data support a model in which RA regulates ovarian follicle development by stimulating granulosa cell proliferation and that this stimulatory effect is at least in part driven by the modulation of Ca2+ signaling.

Gene expression profiling reveals Cyp26b1 to be an activin regulated gene involved in ovarian granulosa cell proliferation.

Activin, a member of the TGF-ß superfamily, is an important modulator of FSH synthesis and secretion and is involved in reproductive dysfunctions and cancers. It also regulates ovarian follicle development. To understand the mechanisms and pathways by which activin regulates follicle function, we performed a microarray study and identified 240 activin regulated genes in mouse granulosa cells. The gene most strongly inhibited by activin was Cyp26b1, which encodes a P450 cytochrome enzyme that degrades retinoic acid (RA). Cyp26b1 has been shown to play an important role in male germ cell meiosis, but its expression is largely lost in the ovary around embryonic d 12.5. This study demonstrated that Cyp26b1 mRNA was expressed in granulosa cells of follicles at all postnatal developmental stages. A striking inverse spatial and temporal correlation between Cyp26b1 and activin-ßA mRNA expression was observed. Cyp26b1 expression was also elevated in a transgenic mouse model that has decreased activin expression. The Cyp26 inhibitor R115866 stimulated the proliferation of primary cultured mouse granulosa cells, and a similar effect was observed with RA and activin. A pan-RA receptor inhibitor, AGN194310, abolished the stimulatory effect of either RA or activin on granulosa cell proliferation, indicating an involvement of RA receptor-mediated signaling. Overall, this study provides new insights into the mechanisms of activin action in the ovary. We conclude that Cyp26b1 is expressed in the postnatal mouse ovary, regulated by activin, and involved in the control of granulosa cell proliferation.

Use of reporter genes to study the activity of promoters in ovarian granulosa cells.

Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and beta-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subunit- and estrogen receptor alpha (ERalpha)-promoter activities. We have demonstrated that estrogen suppresses activin subunit gene promoter activity while activin increases ERalpha promoter activity and increases functional ER activity, suggesting a reciprocal regulation between activin and estrogen signaling in the ovary. We also discuss more broadly some key considerations in the use of reporter genes and cell-based transfection assays in endocrine research.

Activin regulates estrogen receptor gene expression in the mouse ovary.

Activin, a member of the transforming growth factor-beta superfamily, is an important modulator of follicle-stimulating hormone synthesis and secretion in the pituitary and plays autocrine/paracrine roles in the regulation of ovarian follicle development. From a microarray study on mouse ovarian granulosa cells, we discovered that the estrogen receptor beta (ERbeta) is inducible by activin. We previously demonstrated that estrogen suppresses activin gene expression, suggesting a feedback relationship between these two follicle-regulating hormones. The purpose of this study was to investigate fully activin A regulation of ER expression. Real time reverse transcription-PCR assays on cultured granulosa cells showed that both ERalpha and ERbeta mRNAs were induced by activin A at 4, 12, and 24 h in a dose-responsive manner. Western blots confirmed an increase in their protein levels. Consistent with increased ERalpha and ERbeta expression, activin A stimulated estradiol-induced estrogen response element promoter activity. Activin A stimulation of ER expression was a direct effect at the level of gene transcription, as it was not abolished by cycloheximide but was abolished by actinomycin D, and in transfected granulosa cells activin A stimulated ERalpha promoter activity. To investigate the effect of activin in vivo and, thus, its biological significance, we examined ER expression in inhibin transgenic mice that have decreased activin expression and discovered that these mice had decreased ERalpha and ERbeta expression in the ovary. We also found that ER mRNA levels were decreased in Müllerian inhibiting substance promoter (MIS)-Smad2 dominant negative mice that have impaired activin signaling through Smad2, and small interfering RNAs targeting Smad2 or Smad3 suppressed ERalpha promoter activation, suggesting that Smad2 and Smad3 are involved in regulating ER levels. Therefore, this study reveals an important role for activin in inducing the expression of ERs in the mouse ovary and suggests important interplay between activin and estrogen signaling.

Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary.

In the ovary, the steroid hormone estrogen and the TGF-beta superfamily member activin are both produced by granulosa cells and they both have intraovarian functions. Emerging evidence has indicated an interaction of these two signaling pathways. Based on the fact that estrogen and activin can impact early follicle formation and development, we hypothesize that estrogen treatment may alter activin signaling in the neonatal ovary. Therefore, this study was designed to examine the effect of neonatal diethylstilbestrol (DES) and estradiol (E(2)) exposure on the mRNA and protein levels of the key factors involved in activin signaling in the mouse ovary. CD-1 mouse pups were given daily injections of DES, E(2), or oil on postnatal d 1-5, and ovaries and sera were collected on d 19. Neonatal DES or E(2) exposure decreased the number of small antral follicles, induced multioocytic follicle formation, and decreased activin beta-subunit mRNA and protein levels. Consistent with local loss of beta-subunit expression, the phosphorylation of Smad 2, a marker of activin-dependent signaling, was decreased in the estrogen-treated ovaries. The decreased beta-subunit expression resulted in a decrease in serum inhibin levels, with a corresponding increase in FSH. Estrogen also suppressed activin subunit gene promoter activities, suggesting a direct transcriptional effect. Overall, this study demonstrates that activin subunits are targets of estrogen action in the early mouse ovary.

Postnatal regulation of germ cells by activin: the establishment of the initial follicle pool.

Mammalian females enter puberty with follicular reserves that exceed the number needed for ovulation during a single lifetime. Follicular depletion occurs throughout reproductive life and ends in menopause, or reproductive senescence, when the follicle pool is exhausted. The mechanisms regulating the production of a species-specific initial follicle pool are not well understood. However, the establishment of a follicular reserve is critical to defining the length of reproductive cyclicity. Here we show that activin A (rh-ActA), a known regulator of follicle formation and growth in vitro, increased the number of postnatal mouse primordial follicles by 30% when administered to neonatal animals during the time of germline cyst breakdown and follicle assembly. This expansion in the initial follicle pool was characterized by a significant increase in both germ cell and granulosa cell proliferation. However, the excess follicles formed shortly after birth did not persist into puberty and both adult rh-ActA- and vehicle-treated animals demonstrated normal fertility. A follicle atresia kinetic constant (k(A)) was modeled for the two groups of animals, and consistent with the empirical data, the k(A) for rh-ActA-treated was twice that of vehicle-treated animals. Kinetic constants for follicle formation, follicle loss and follicle expansion from birth to postnatal day 19 were also derived for vehicle and rh-ActA treatment conditions. Importantly, introduction of exogenous rh-ActA revealed an intrinsic ovarian quorum sensing mechanism that controls the number of follicles available at puberty. We propose that there is an optimal number of oocytes present at puberty, and when the follicle number is exceeded, it occurs at the expense of oocyte quality. The proposed mechanism provides a means by which the ovary eliminates excess follicles containing oocytes of poor quality prior to puberty, thus maintaining fertility in the face of abnormal hormonal stimuli in the prepubertal period.

Estradiol and testosterone have opposite effects on microtubule polymerization.

We have reported earlier the purification of tubulin from a plasmalemmal-microsomal fraction derived from rat hippocampus using an estradiol (E(2)) affinity column and the specific binding of tubulin to both E(2) and testosterone (T). To further investigate the effect of E(2) and T on the function of this protein, changes in microtubule polymerization as a result of exposure to the steroids were examined in this study, using both pure tubulin and rat hippocampal primary cell cultures. First, pure tubulin was incubated with or without steroids for 30 min on ice followed by polymerization at 37 degrees C. The numbers of microtubules formed were counted from electron microscopic pictures. The results showed that at 30 min of polymerization, 10 nM, 30 nM and 30 microM of E(2) inhibited microtubule assembly by -70%, -94%, and -92%, respectively (p < 0.01), while T at the same three concentrations stimulated it by +83%, +66%, and +121%, respectively (p < 0.05). The inhibitory effect of E(2) and the stimulatory effect of T were observed at 15, 30 and 60 min of the polymerization process. Next, primary cell cultures from 17-day rat fetus hippocampal tissues were treated with the steroids and polymerized microtubules (Triton X-100 resistant) were examined by immunocytochemistry. The results demonstrated that 60 min of E(2) treatment (10 nM) decreased the intensity of the immunolabeling of polymerized microtubules. The effect of T at nM concentration was not significant though it increased the immunolabeling at microM concentration. Of great significance was a remarkable inhibition by T of the well-established depolymerization effect of colchicine in both the pure tubulin assay and the cell culture model, while E(2) was not effective. In an effort to pursue the possible mechanism(s) of the effect of E(2) and T on microtubule formation, we found that T only inhibited the microtubule depolymerization process without affecting the rate of polymerization. In contrast, E(2) modifies only the polymerization process without altering the depolymerization. Overall, these data indicate that E(2) and T may be considered as novel regulators of microtubule dynamics and thereby controlling cytoskeleton function in cells.