Antibacterial Activity of Ag‐Hydroxyapatite Coating Against Hematogenous Infection by Methicillin‐Resistant Staphylococcus aureus in the Rat Femur

Several antibacterial materials have been developed to prevent periprosthetic joint infection and thus prevent serious complications for patients and surgeons. However, no study has addressed the activity of antibacterial materials against hematogenous infection. The present study evaluated the antibacterial activity of a silver‐containing hydroxyapatite‐coated implant against methicillin‐resistant Staphylococcus aureus (MRSA) hematogenous infection. Implants coated with hydroxyapatite and silver‐hydroxyapatite were inserted into rats’ right and left femurs, respectively, after which the animals were infected with S. aureus via a tail vessel. About 10⁷ colony‐forming units was the optimal bacterial number for the establishment of S. aureus hematogenous infection. Bacterial loads and C‐reactive protein in the blood were measured to confirm bacteremia and inflammation. Fourteen days after the infection, bacterial loads were statistically lower in the femurs containing silver‐hydroxyapatite‐coated implants than in those with hydroxyapatite‐coated implants (p = 0.022). Thus, silver‐hydroxyapatite‐coated implants might provide antibacterial activity against MRSA hematogenous infection in the postoperative period. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2655–2660, 2019     

Insect’s intestinal organ for symbiont sorting

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host–symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ’s ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host’s control and symbiont’s motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.Diverse organisms are obligatorily associated with microbial symbionts, which significantly contribute to their adaptation and survival (1–3). In such symbiotic associations, the host organisms often develop specialized cells, tissues, or organs for harboring their specific microbial partners [for example, root nodules in the legume–Rhizobium symbiosis (4, 5), symbiotic light organs in the squid–Vibrio symbiosis (6, 7), and bacteriocytes in the aphid–Buchnera symbiosis (8, 9)].These microbial symbionts are either acquired by newborn hosts from the environment every generation as in the legume–Rhizobium and the squid–Vibrio symbioses or transmitted vertically through host generations as in the aphid–Buchnera symbiosis (10). In the environmentally acquired symbiotic associations, it is essential for the host organisms to recognize and incorporate specific symbiotic bacteria while excluding a myriad of nonsymbiotic environmental microbes (6, 11). In the vertically transmitted symbiotic associations, it is important for the host organisms to selectively transmit their own symbiotic bacteria while excluding parasitic/cheating microbial contaminants (12–14). Hence, it is expected that the host organisms must have evolved some mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Those controlling mechanisms are of general importance for understanding symbiosis (6, 10).Stinkbugs, belonging to the insect order Hemiptera, consist of over 40,000 described species in the world (15). The majority of the stinkbugs suck plant sap or tissues, and some of them are notorious as devastating agricultural pests (16). These plant-sucking stinkbugs possess a specialized symbiotic organ in their alimentary tract: A posterior region of the midgut is morphologically differentiated with a number of sacs or tubular outgrowths, called crypts or ceca, whose inner cavity hosts symbiotic bacteria (17–21). Usually, a single bacterial species dominates in the midgut crypts, and elimination of the symbiont causes retarded growth and increased mortality of the host, which indicates the specific and beneficial nature of the stinkbug gut symbiosis (20–31). The initial symbiont infection is established by nymphal feeding, which may be either via vertical transmission from symbiont-containing maternal secretion supplied upon oviposition (19–21) or via environmental acquisition from ambient microbiota (21–23). What mechanisms underlie the selective establishment of a specific bacterial symbiont in the midgut symbiotic organ despite the oral inoculum contaminated by nonsymbiotic microbes has remained largely an enigma, although recent studies have started to shed light on the symbiotic mechanisms underlying the environmental acquisition of specific Burkholderia symbionts in the bean bug Riptortus pedestris (Hemiptera: Alydidae) (22, 32). Antimicrobial substances produced by the midgut epithelia (33, 34) and some symbiont factors, such as stress-responsive polyester accumulation, cell wall synthesis, and purine biosynthesis (35–37) might be involved in the selective infection of the Burkholderia symbiont to the midgut crypts.Here we address this important symbiotic issue by the discovery of a previously unrecognized intestinal organ in the stinkbugs. Though very tiny and inconspicuous, the organ governs the configuration and specificity of the stinkbug gut symbiosis. Lying in the middle of the midgut, the organ blocks food flow and nonsymbiotic bacteria but selectively allows passing of specific symbiotic bacteria, whereby the stinkbug’s intestine is functionally partitioned into the anterior region specialized for digestion and absorption and the posterior region dedicated to symbiosis. The blocking of food flow by the organ is conserved across diverse stinkbug families, suggesting the possibility that the organ evolved in their common ancestor and has played substantial roles in their symbiont-mediated adaptation and diversification.     

Decreased levels of covalently bound ceramide are associated with ultraviolet B-induced perturbation of the skin barrier

Although ultraviolet B (UVB) irradiation perturbs the skin barrier, little is known about the mechanism(s) with respect to the metabolism of ceramide (Cer). We examined changes in intercellular lipids in murine stratum corneum following UVB irradiation. A single UVB (75 mJ per cm(2)) irradiation caused a significant increase in transepidermal water loss, which plateaued at day 4. In parallel, covalently bound Cer was significantly decreased with the greatest decrease at days 3-4. In contrast, the levels of other free, non-bound lipids (including Cer or acylceramides) were significantly increased for Cer, or remained unchanged at day 4 compared with non-irradiated controls. RT-PCR analysis demonstrated a significant decrease in mRNA encoding transglutaminase-1 (TGase1). The peak occurred 2-4 d after a single UVB irradiation, a time when covalently bound Cer was significantly downregulated in concert with the disruption of the skin barrier. Furthermore, UVB-induced epidermal hyperplasia occurred to the greatest extent between 2 and 4 d following UVB irradiation. These results suggest that decreases in covalently bound Cer in the stratum corneum are mediated via the downregulation of TGase-1 as well as by the rapid induction of epidermal hyperplasia, which is attributable to the perturbation of the skin barrier induced by UVB irradiation.     

Long-term alteration in the expression of keratins 6 and 16 in the epidermis of mice after chronic UVB exposure

The influences of chronic UVB exposure on epidermal differentiation have been poorly studied compared to dermal photo-aging although those effects are very important in terms of photo-damage to the skin. The purpose of this study was to investigate the effects of chronic UVB exposure on keratin expression in the epidermis. The effects on murine skin of chronic exposure to weak UVB (below 1 MED) was examined by immunoblotting for keratins K10, K5, K6, and K16, by immunohistochemistry using antibodies to K6, K16, and Ki67 as well as by conventional HE staining of skin sections. Alterations of keratin expression induced by the chronic UVB exposure were distinct from those elicited by a single acute UVB exposure. The expression of keratins K6 and K16 was quite long-lasting, continuing for 7 weeks after 6 weeks of chronic UVB exposure and for 6 weeks after 9 weeks of chronic UVB exposure. In contrast, K6 and K16 expression induced by a single UVB exposure at 0.5 MED or 3 MED almost ceased within 2 weeks after that exposure. Furthermore, the expression of the constructive keratins, K5 and K10, remained almost unchanged by chronic UVB exposure. Epidermal thickness was increased significantly immediately after the 9 weeks of chronic UVB exposure; however, it had returned to normal level 6 weeks later. The alterations in keratin expression accompanied the marked disruption of the ordered ultrastructure of keratin intermediate filaments, which were observed by TEM. Thus, chronic exposure to UVB has a deep impact on the biosynthetic regulation of different keratins in the epidermis, thereby interfering with the ordered ultrastructure of keratin intermediate filaments. Those events could have relevance to the mechanism of photo-damage, such as fine wrinkles observed in chronically UV-exposed skin in addition to dermal photo-aging.     

Inhibition of ultraviolet-B-induced wrinkle formation by an elastase-inhibiting herbal extract: implication for the mechanism underlying elastase-associated wrinkles

BACKGROUND: Previously, we have demonstrated that fibroblast-derived elastase plays an essential role in the increased three-dimensional tortuosity of elastic fibers, contributing to the loss of skin elasticity in UV-B-exposed skin. This decrease in skin elasticity is closely associated with the formation of wrinkles induced by UV exposure. OBJECTIVE: To further clarify the role of elastase in the formation of wrinkles induced by UV exposure, we assessed the effects of an extract of Zingiber officinale (L.) Rose (which inhibits fibroblast-derived elastase) on the wrinkle formation induced by chronic UV-B irradiation. RESULTS: Topical application of an extract of Zingiber officinale (L.) Rose to rat or hairless mouse skin significantly inhibited the wrinkle formation induced by chronic UV-B irradiation at a suberythemal dose, which was accompanied by a significant prevention of the decrease in skin elasticity in both types of animal skin. In the rat hind limb skin, consistent with the inhibition of reduced skin elasticity, wrinkle prevention occurred concomitantly with a significant decrease in the curling and three-dimensional tortuosity of dermal elastic fibers. CONCLUSION: Our results indicate that herbal extracts with an ability to inhibit fibroblast-derived elastase may prove to be effective as anti-wrinkling agents, confirming the important role of elastase in UV-B-induced wrinkle formation.     

Evaluation of unique elastic aggregates (elastic globes) in normal facial skin by multiphoton laser scanning tomography

Background

There is no reliable marker to estimate the degree of skin aging in vivo. It now has become possible to quantitatively determine the dermal characteristics of the extracellular matrix (ECM) in vivo using multiphoton laser tomography (MLT).

Methods

Fifty-seven healthy Japanese female volunteers, aged from 20 to 60 years old, were examined using multiphoton depth-resolved measurements of autofluorescence (AF) and second harmonic generation (SHG) at three sites on their right cheek. Paraffin-embedded skin specimens obtained from the faces of 12 normal individuals aged 38-68 years old were stained with Elastica van Gieson (EVG).

Results

We found unique elastic aggregates at a 20µm depth from the dermo-epidermal junction (DEJ) in vivo which increased in size with aging of subjects from 20 to 60 years old. SHG fibers seemed to surround those elastic aggregates. Histological examination of specimens from normal individuals stained with EVG confirmed the occurrence of elastic aggregates with varied sizes just beneath the epidermis or hair follicles.

Conclusions

The elastic aggregates are morphologically similar to previously described ‘elastic globes’ and can serve as a marker of the early stage of photoaging. MLT will contribute to determine age-related dermal changes using a non-invasive technique.

     

Expression of uncoupling proteins in human skin and skin-derived cells

Uncoupling protein (UCP) is a mitochondrial membrane protein that uncouples oxidative phosphorylation. The physiological function of major isoforms of UCPs is related to the control of body temperature and reactive oxygen species production. Although skin is an important organ for heat radiation and protection against stress, the expression and function of UCPs in the skin have remained unclear. The expression of UCPs in human skin and its derived cells was researched at the mRNA and protein levels. The effects of norepinephrine (NE) and 9-cis retinoic acid (RA) on UCP expression were also investigated. The expression of UCP1 mRNA was found in the human epidermis and was upregulated in differentiated keratinocytes. UCP1 expression in keratinocytes was synergistically upregulated by NE and RA treatment. Significant expression of UCP2 and UCP3 was observed also in cultured keratinocytes and fibroblasts. By immunohistochemistry, localization of UCP1 was found in the granular layer of the epidermis, sweat glands, hair follicles, and sebaceous glands of various sites in the human body. UCP3 was widely found in the dermis. This showed that UCPs exist in human skin, with their expression being under hormonal control. These findings are in stark contrast with the well-accepted view of UCP1 expression being exclusive to brown adipose tissue.