ObjectiveTo evaluate the survival and function of hepatocytes (HCs) on a novel three-dimensional (3D) synthetic biodegradable polymer scaffold with an intrinsic network of interconnected channels under continuous flow conditions. Summary Background DataThe authors' laboratory has investigated HC transplantation using 3D biodegradable polymers as scaffolding as an alternative approach to treatment of end-stage liver disease. Previous studies have demonstrated survival of HCs transplanted on polymer discs in peripheral tissue sites and partial correction of single enzyme liver defects. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells; this is thought to be caused by inadequate oxygen diffusion. MethodsHCs and nonparenchymal liver cells from Lewis rats were seeded onto 3D biodegradable polymer scaffolds. Microporous 3D polymers were created using 3D printing on copolymers of polylactide-coglycolide. The cell/polymer constructs were placed in static culture or continuous flow conditions. The devices were retrieved after 2 days and examined by scanning electron microscopy and histology. Culture medium was analyzed for albumin by enzyme-linked immunosorbent assay (ELISA). Differences in culture parameters including pH, PCO2, P02, glucose, lactate, and HCO3 were examined. ResultsScanning electron microscopy revealed successful attachment of HCs on the 3D poymer in both static and flow conditions. Histology demonstrated viable HCs in both conditions. EUSA demonstrated a significantly higher mean concentration of albumin in flow conditions than in static conditions. Cufture parameter analysis revealed a significantly higher P02 and glucose level, and a more physiologic pH in flow conditions than in static conditions. ConclusionsHCs cocultured with nonparenchymal cells can attach to and survive on the 3D polymer scaffolds in both static and flow conditions in the size and configuration used in this study. Flow conditions may provide a more conducive environment for HC metabolism and albumin synthesis than static conditions. The authors hypothesize that flow through directed channels will be necessary for the transfer of large masses of cells when implantation studies are initiated.Each year 26,000 people die of end-stage liver disease in the United States, with an estimated annual cost of $9

We have isolated cDNAs from maize (ZGB1) and Arabidopsis (AGB1) encoding proteins homologous to P subunits of guanine nucleotide-binding protein (G protein (9,10) or the transmission of red and blue light-induced signals (11,12). Furthermore, the cloning ofArabidopsis gene GPAI encoding a G protein a subunit designated GPal and its tomato homologue has provided molecular evidence for the existence of G protein-mediated signaling pathway(s) in plants (13, 14). The reports of G protein involvement in a number of different cellular functions raise the possibility that GPal may be involved in more than one signaling pathway. It is possible that G protein f3 subunit(s) might also play a direct role in signaling in plants.In this report, we describe the cloning of the maize ZGB1 and Arabidopsis AGB1 cDNAs encoding proteins that share >41% identity with animal G protein f3 subunits. § This level of homology is greater than that between a known yeast G protein (3subunit and animal ones (15). ZGB1 and AGB1 may represent an additional type of G protein f subunit that is conserved in flowering plants and expressed in roots, leaves, and flowers. MATERIALS AND METHODSMaize Subtracted Library. A maize tassel cDNA library in pCDNAII (Invitrogen) and a maize ear shoot cDNA library in A-Uni-ZAP (Stratagene) were provided by M. Albertsen and G. Huffman, respectively, of Pioneer Hi-Bred International. Biotinylated RNA was generated in vitro from the ear shoot library by using the Gemini Riboprobe system (Promega) with the manufacturer's protocol modified to include 1 mM biotin-11 rUTP (Enzo Diagnostics, instead of photobiotin rUTP) and an increased rUTP concentration (1 mM) in the transcription reaction. A subtracted tassel cDNA library was prepared by hybridization of the biotinylated RNAs with single-stranded DNA from the tassel library as described (16). The second strand of the nonsubtracted DNA molecules was synthesized with Klenow as described (17). One of the obtained clones was designated pPHP2541.S' Rapid Amplification of cDNA Ends (RACE) for PCR. 5' RACE primer extension was performed by using the 5' RACE system (GIBCO/BRL) with leaf and tassel poly(A)+ RNA and the oligonucleotide 5'-GATATCCACAGCCTA-CAGTTG-3' derived from the sequence of the pPHP2541 cDNA insert. The pPHP2541-derived nested primer 5'-GTATTTGATGAGTTGATGGAC-3' and the provided anchor primer were used for PCR amplification with Taq I polymerase (Perkin-Elmer). A clone containing a 0.6-kb PCR product was named pPHP3573.Library Screening, Subcloning, and Sequence Analysis. A A-Uni-ZAP maize tassel cDNA library was screened for a full-length cDNA by using standard conditions and a labeled 0.9-kb insert from pPHP2541 as a probe. A AYES cDNA library from Arabidopsis thaliana (ref. 18; a gift from J. Mulligan) was screened with 32P-labeled 0.9-kb and 0.6-kb maize cDNAs from pPHP2541 and pPHP5373, respectively. cDNAs were subcloned into the Promega vector pGEM7Zf(+) for sequencing of both strands.Southern and Northern Blot Hybridizations. Genomic DNA isol...

The swabbing and tape-stripping methods have traditionally been used for collecting skin microbiome samples for skin bacterial analysis, although no reports have compared the outcome of these methods for collecting skin bacteria. Our purpose was to show the differences in microbial composition between samples collected using the swabbing and tape-stripping methods, by both the next generation sequencing and culture studies. The skin microbiome was collected by both methods, and the samples were processed for a sequence-based microbiome analysis and culture study. The next-generation sequencing results showed that skin bacteria collected using the tape-stripping method were comparable to those collected using the swabbing method. In the culture study, the tape-stripping method collected a greater number and wider variety of viable skin bacteria than the swabbing method. These results suggest that the tape-stripping method is comparable to the swabbing method for collecting viable skin bacteria, without losing fidelity to the composition of skin microbiome.

Estrogen promotes cutaneous wound healing in ovariectomized (OVX) female mice. However, the effects of topical estrogen application on wounds remain unclear. Therefore, the aim of this study was to compare the effects of topical estrogen application on wounds with standard treatment methods. Eight-week-old C57BL/6J female mice underwent OVX and received two full-thickness wounds four weeks later. Mice were divided into three groups: topical estradiol benzoate (EB) (0.75 μg/g/day) wound treatment, subcutaneous estradiol (E2) pellets (0.05 mg, 21 days), and topical E2 (0.01 g/day) skin application. Wound healing was observed until day 14. Wound area ratios were significantly smaller in the topical EB wound treatment group than in the subcutaneous E2 pellet group on days 1–14 (p < 0.05) and topical E2 skin application group on days 1–9 (p < 0.05). Neutrophil and macrophage numbers were significantly smaller in the topical EB wound treatment group than in the subcutaneous E2 pellet and topical E2 skin application groups on day 7 (p < 0.05). Moreover, the number of new blood vessels and ratio of myofibroblasts were significantly larger in the topical EB wound treatment group than in the subcutaneous E2 pellet and topical E2 application skin groups on day 7 (p < 0.05). These results demonstrate that the application of estrogen to wounds reduced inflammatory responses and promoted angiogenesis and wound contraction more than the two other standard treatment methods.

With the increase in the older populations, the number of bedridden older patients is becoming a matter of concern. Skin microbiome and skin physiological functions are known to change according to lifestyle and community; however, such changes in case of movement-and cleaning-restricted bedridden older patients have not yet been revealed. To address this issue, we analyzed skin microbiome and skin physiological functions, including pH, hydration, sebum level, and transepidermal water loss (TEWL), of bedridden older patients, compared with those of ambulatory older and young individuals. For this analysis, we enrolled 19 healthy young and 18 ambulatory older individuals from the community and 31 bedridden older patients from a single, long-term care hospital in Japan. The area of interest was set to the sacral (lower back) skin, where pressure injuries (PIs) and subsequent infection frequently occurs in bedridden older patients. We observed a higher number of gut-related bacteria, fewer commensals, higher skin pH, and lower TEWL on the sacral skin of bedridden older patients than on that of young or ambulatory older individuals. In addition, we observed that 4 of the 31 bedridden older patients developed PIs during the research period; a higher abundance of pathogenic skin bacteria were also observed inside the PI wounds. These findings imply distinct skin microbiome and skin physiological functions in bedridden older patients in comparison with healthy individuals and may suggest the need for more stringent cleaning of the skin of bedridden older patients in light of the closeness of skin and wound microbiome.

We investigated cold plasma effects on acute wounds of mice. The mice were classified into experimental and control groups. In the former, wounds were treated using cold plasma once daily for 1 minute, and then covered with hydrocolloid dressing; wounds in the control were left to heal under hydrocolloid dressing. Daily evaluation was conducted for 15 days. General and specific staining was applied to evaluate re-epithelialization, neutrophil, macrophage, myofibroblast and transforming growth factor beta. It was found that cold plasma accelerated wound healing by one day. Plasma may promote the late phase of inflammation, accelerate reepithelialization and increase wound contraction.3

It is becoming increasingly important for clinicians to identify a safer intramuscular (IM) injection site in the deltoid muscle because of possible complications following the vaccine administration of IM injections. We herein examined 4 original IM sites located on the perpendicular line through the mid-acromion to establish a safer IM injection site. Thirty healthy volunteers participated in this study and the distances from our 4 IM sites to some anatomical landmarks on their left arms were measured. Ultrasonography (US) was also performed to measure the thickness of the deltoid muscle and identify the posterior circumflex humeral artery (PCHA) along the course of the axillary nerve. Subcutaneous thickness was measured using 2 methods: measuring the skin thickness with caliper after pinching the skin, and with US. The results obtained revealed that the intersection between the anteroposterior axillary line (the line between the upper end of the anterior axillary line and the upper end of the posterior axillary line) and the perpendicular line from the mid-acromion was the most appropriate site for IM injections because it was distant from the axillary nerve, PCHA, and subdeltoid/subacromial brusa. At this site, depth of needle insertions was 5 mm greater than the subcutaneous thickness at a 90° angle, which was sufficient to penetrate subcutaneous tissue in both sexes. Subcutaneous thickness can be assessed with almost the same accuracy by US or measuring with calipers after pinching the skin. The results of the present study support the improved vaccine practice for safer IM injections.

Since lymphedema rarely develops in the mouse hindlimb, the underlying mechanisms remain unclear. We herein investigated the resolution of chronic hindlimb lymphedema in mice using a Near-Infrared Fluorescence (NIRF) imaging system. Nineteen 7–28-week-old BALB/c male and female mice were injected with two dyes for lymphography and dissection. Lymphadenectomy was performed on six male mice to completely obstruct lymph flow in the hindlimb. Edematous changes in both hindlimbs were compared until 60 days after surgery. The NIRF imaging system detected three lymphatic collecting systems in the mouse hindlimb: superficial lateral, superficial medial, and deep medial. It also showed connections between the superficial and deep lymphatic systems in the inguinal region. Lymphadenectomy of the iliac, inguinal, and popliteal lymph nodes caused edematous changes. However, lymph flow in these operated areas restarted within 60 days and the severity of lymphedema appeared to be low. NIRF imaging showed that the deep medial system and a connection between the superficial and deep lymphatic systems in the inguinal region drain lymph from the hindlimb. This is the one reasons why lymphedema does not develop in the mouse hindlimb. The stable obstruction of lymph flow in these three systems is desired to develop chronic lymphedema.