Unwounded Control Research Articles

First report of walnut anthracnose caused by Colletotrichum orientalis in South Korea.

Walnut (Juglans regia L.) is known as "hodu" in South Korea. It is cultivated as a cash crop and an ornamental plant across South Korea. Walnut anthracnose is one of the main limiting factors for walnut production. In June 2022, severe anthracnose was observed in several walnut fields in Gimcheon, South Korea. The disease incidence was ~35% and symptoms on affected fruits consist of necrotic and circular lesions with or without conidial mass. Six pure isolates with similar morphological characteristics were obtained from the tissue of six affected fruits. Diseased tissues were cut into 3-to-4 mm2 pieces, surface sterilized by 1% sodium hypochlorite (NaOCl) for 2 minutes, and washed three times in sterile distilled water, then dried by blotting. Four pieces/plates were placed onto potato dextrose agar (PDA; Difco Becton Dickinson) and incubated at 25°C in the dark for 2-3 days. To obtain pure isolates, hyphal tips were transferred onto fresh PDA. The colonies of all isolates on the PDA were light gray to pinkish on the upper side and pink to vinaceous on the reverse after 7 days of incubation at 25°C in the dark. The conidia were hyaline, fusiform, aseptate, and 11.3 to 20.1 µm × 3.6 to 6.9 µm (n =100). Appressoria were single, cylendrical, smooth-walled, dark brown, and 8.1 to 14.3 × 5.4 to 10.5 μm (n =30). The morphological characteristics of the isolates were comparable with those of Colletotrichum orientalis (Chen et al. 2022). Three isolates (WN23, WN28 and WN30P) were selected based on morphological characteristics for molecular identification. The beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and histone 3 (HIS3) genes of selected isolates were amplified and sequenced using the primers Bt2a/Bt2b, ACT512F/ACT783R, CHS-79F/CHS-345R and CYLH3F + CYLH3R, respectively (Damm et al. 2012). The resulting consensus sequences of each gene were deposited in GenBank with the accession numbers (TUB2: LC742910 to LC742912; CHS-1: LC742916 to LC742918; ACT: LC742919 to LC742921, HIS3: LC742922 to LC742924). The constructed maximum likelihood tree based on TUB2, ACT, CHS-1, and HIS3 sequence data revealed that the three isolates formed a clade with C. orientalis reference strain CBS 128532 (ex-type). The pathogenicity of two representative isolates (WN28 and WN30P) was confirmed on healthy, surface-sterilized, and detached walnut fruits. Both wounded and nonwounded fruits (10/isolate) were inoculated with the conidial suspension (106 spores/ml). The wounded and unwounded control fruits were inoculated with sterile distilled water. All the fruits (inoculated and control) were incubated in containers at 25°C with a 12-h/12-h light/dark cycle. This experiment was repeated twice. Necrotic lesions were observed in all inoculated wounded fruits, while unwounded and control fruits remained asymptomatic. The pathogen was re-isolated from inoculated fruits and identified as C. orientalis by morphological characteristics and ITS and TUB2 sequences. Damm et al. (2012) has identified C. fioriniae as species belongs to two well separated clades. However, Chen et al. (2022) split the C. fioriniae species into two species, with the new species called C. orientalis, as a causal agent of apple bitter rot. In previous studies, C. fioriniae has been reported as a causal of walnut anthracnose (Luongo et al. 2021; Zhu et al. 2015; Varjas et al. 2019). To our knowledge, this is the first report of C. orientalis as the causal agent of walnut anthracnose in South Korea. Since this disease reduces the commercial value and quality of walnut fruits, effective management practices should be developed to deal with C. orientalis.

First Report of Brown Leaf Spot Caused by Epicoccum tobaicum on Oat (Avena sativa) in Korea.

Oat (Avena sativa) is one of the Korean winter crops, and oat consumption has been increasing because it is widely perceived as a superfood. Recently, various fungal diseases have been reported likely due to climate changes during the winter season in Korea (Choi et al., 2018; Kim, 2020). During the 2020-2021 winter to spring, we surveyed new fungal diseases among cereals, including oats, in the southern region of Korea. In April 2021, brown leaf spots on oat leaves were observed in Gangjin, Jeollanam-do, Korea. These brown spots were irregular circles, ranging from 2-7 mm in diameter. Samples from three infected leaves were surface sterilized by treating them with 70% ethanol for 1 min and 1% NaOCl for 1 min. The samples were subsequently rinsed at least twice with distilled water and dried with a sterile paper towel before being placed on 1.5% water agar supplemented with 100 ppm streptomycin. Hyphal tips derived from infected tissues after incubation at 25C for 7 days were transferred to a fresh potato dextrose agar (PDA). Three isolates, labeled as KJO-AN2-S1, KJO-AN2-S2 and KJO-AN2-S3, were obtained via single hyphal tip purification. Colonies on PDA were pigmented vermilion and subsequently turned to saffron color with irregular margins after 7 days. Conidia produced on PDA were golden to dark brown, globose to subglobose, solitary, and measured 15.5-21.5 μm in diameter (n=50). Cultural and morphological characteristics suggested that these isolates belong to Epicoccum species (Chen et al. 2017). For identification by sequencing, the ITS (MW691866-68), tub2 (MW691872-74), and rpb2 (MW691869-71) regions of three isolates were amplified using the primer pairs ITS5/ITS4 (White et al., 1990), Btub2Fd/ Btub4Rd (Woudengerg et al., 2009), and RPB2-5F2 (Sung et al., 2007)/fRPB2-7cR (Liu et al., 1999), respectively. A maximum likelihood phylogenetic analysis based on the concatenated ITS, tub2, and rpb2 sequences placed the three isolates within a clade comprising E. tobaicum CBS 384.36. A mycelial plug (5 mm diameter) was inoculated onto wounded and unwounded leaves of healthy 12-day-old oat (cv. Choyang) seedlings. The control leaves were inoculated with a sterile PDA plug. All inoculated and control plants were placed in a plastic box and incubated at 20℃ in darkness with 100% humidity. After 1 day, the inoculated mycelial plug or sterile PDA plug from plants was removed; the plants in plastic boxes were then transferred to a growth chamber set at 20℃ with 12 h light and 60-70% humidity. While brown spot lesions were observed in both unwounded and wounded leaves 7 days post-inoculation, both wounded and unwounded control leaves remained asymptomatic. The pathogen was recovered from all symptomatic leaf tissues but could not be isolated from control leaves. The re-isolated pathogen was identified as E. tobaicum through morphological characterization and sequence-based identification, fulfilling Koch's postulates. This study is the first to report a causal relationship between E. tobaicum and brown leaf spot disease of oat in Korea. Identification of this newly emerging fungal disease on oats will help prepare for effectively managing this disease.

First Report of Anthracnose on Chili Pepper Caused by Colletotrichum sojae in Hebei Province, China.

China is the largest chili pepper producing country, and Hebei Province stands out as the forth with planting area at about 1500 km2 in China. Pepper (Capsicum annuum L.) is susceptible to Colletotrichum spp. infection during its growth, which seriously affects production yield and quality. In September 2020, widespread anthracnose was observed on pepper in Hebei (115.48° N, 38.77° E), China. Necrotic lesions on pepper fruits were suborbcular, sunken, with acervuli arranged in the middle of lesion (e-Xtra 1A). To perform fungal isolation, small tissue with 0.3 cm2 in size at the symptomatic tissue margin was surface disinfested with 75% ethanol for 10 s, and 0.1% HgCl2 for 40 s, then washed three times with sterile ddH2O. Fragments were placed on potato dextrose agar (PDA) amended with 100 mg·L-1 chloramphenicol and incubated at 28 ºC under darkness for 4 days. One of the strains of Colletotrichum spp., named HQY157, was purified by single-spore isolation, then used for morphological characterization, phylogenetic analysis, and pathogenicity tests. Colonies presented light grey aerial mycelium, occasionally mixed with gray-black strips, and the reverse was similar to the surface on PDA (e-Xtra 1B). Conidia were smooth-walled, aseptate, straight with obtuse to slightly rounded ends, 17.3-28.5 × 3.1-7.4 μm (n=50) (e-Xtra 1C). For molecular identification, the internal transcribed spacer (ITS) region, partial sequences of actin (ACT), β-tublin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS) were sequenced using the specific primers (Weir et al. 2012). Sequences were deposited in GenBank with the following accession numbers OM317600-OM317604. A Maximum-Likelihood phylogenetic tree was constructed, based on the concatenated sequences (ACT, CHS, GAPDH, TUB, and ITS) of HQY157 and other closely matching Colletotrichum species obtained from GenBank, by using MEGA-X. It showed that HQY157 was grouped with the C. sojae with bootstrap values of 100% (e-Xtra 2). To confirm the pathogenicity, surface-sterilized healthy pepper fruits and healthy fruits with wounds (deal with a sterile toothpick after surface-sterilized) were then inoculated with 2 μL of conidial suspension (106 conidia/mL). The fruits inoculated with 2 μL sterile distilled water were taken as negative controls. After inoculation, the fruits were kept in a plastic box with sterilized filter paper moistened with sterilized water, and maintained at 25°C in the dark. The experiment was repeated three times. Anthracnose symptoms were observed 7 days after inoculation on the wounded pepper fruits, whereas the unwounded and negative control fruits remained symptomless (e-Xtra 1D). Colletotrichum sojae was re-isolated from the infected pepper fruits and identified by morphological and molecular analysis, fulfilling Koch's postulates. Colletotrichum sojae occurs mainly on Fabaceae plants such as Glycine max, Medicago sativa, Phaseolus vulgaris, and Vigna unguiculata (Damm et al. 2019, Talhinhas and Baroncelli, 2021), and Panax quinquefolium (Guan et al. 2021). To our knowledge, this is the first report of C. sojae causing anthracnose on pepper in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this chili pepper disease.

Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium.

Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese (DiO) mice, a pretype 2 diabetic model. We hypothesize that pannexin1 is dysregulated, and the interaction of two ion-channel proteins (P2X7 and pannexin1) is altered in pretype 2 diabetic tissue. Pannexin1 was found to be present along cell borders in unwounded tissue, and no significant difference was observed between DiO and B6 control. However, an epithelial debridement induced a striking difference in pannexin1 localization. The B6 control epithelium displayed intense staining near the leading edge, which is the region where calcium mobilization was detected, whereas the staining in the DiO corneal epithelium was diffuse and lacked distinct gradation in intensity back from the leading edge. Cells distal to the wound in the DiO tissue were irregular in shape, and the morphology was similar to that of epithelium inhibited with 10Panx, a pannexin1 inhibitor. Pannexin1 inhibition reduced mobilization of calcium between cells near the leading edge, and MATLAB scripts revealed a reduction in cell-cell communication that was also detected in cultured cells. Proximity ligation was performed to determine if P2X7 and pannexin1 interaction was a necessary component of motility and communication. While there was no significant difference in the interaction in unwounded DiO and B6 control corneal epithelium, there was significantly less interaction in the wounded DiO corneas both near the wound and back from the edge. The results demonstrate that pannexin1 contributes to the healing response, and P2X7 and pannexin1 coordination may be a required component of cell-cell communication and an underlying reason for the lack of pathologic tissue migration.

16416 Cutaneous wounds and wound infection affect cognition and behavior in mice

Associated depression and cognitive disorders contribute to poor outcomes in patients with chronic wounds. Here we ask the question: does skin dysbiosis as evidenced by chronic infection in the wound alter cognition and behavior, just as altered microbiota of the gut does? The main goals are to test the hypothesis that wounding and wound infection contribute to cognitive impairment and depressive behaviors in mice with skin wounds infected with Pseudomonas aeruginosa, a common wound pathogen. Wounded, wounded/infected, or unwounded control C57Bl mice were examined using the forced swim test for depressive behavior, the light/dark box test for anxiety, and the novel object recall test for decreased recognition memory. Wounded animals exhibited higher level of depressive behavior, increased anxiety, and decreased cognition (recognition memory) relative to unwounded animals. Animals with infected wounds had similarly increased depressive behavior and anxiety, as well as altered cognition, relative to unwounded animals. We probed the animals’ hippocampi using qPCR for mediators to investigate a possible pathogenic mechanism for the observed behavioral changes. Expression of NOD1, NOD2, (Nod-like receptors that are intracellular sensors of pathogen-associated molecular patterns), NR3C1, NR3C2t (glucocorticoid receptors), and cytokines IL-10 and TNF-α were elevated. These changes were present in the brains of both the wounded and the wounded/infected animals, though the magnitude of change was higher in the wounded/infected animals. Understanding the contribution of the skin-microbiota-brain axis to cognitive function may provide novel approaches to improve outcomes of patients with chronic, infected wounds.

Functional characterisation of the transcriptome from leaf tissue of the fluoroacetate-producing plant, Dichapetalum cymosum, in response to mechanical wounding

Dichapetalum cymosum produces the toxic fluorinated metabolite, fluoroacetate, presumably as a defence mechanism. Given the rarity of fluorinated metabolites in nature, the biosynthetic origin and function of fluoroacetate have been of particular interest. However, the mechanism for fluorination in D. cymosum was never elucidated. More importantly, there is a severe lack in knowledge on a genetic level for fluorometabolite-producing plants, impeding research on the subject. Here, we report on the first transcriptome for D. cymosum and investigate the wound response for insights into fluorometabolite production. Mechanical wounding studies were performed and libraries of the unwounded (control) and wounded (30 and 60 min post wounding) plant were sequenced using the Illumina HiSeq platform. A combined reference assembly generated 77,845 transcripts. Using the SwissProt, TrEMBL, GO, eggNOG, KEGG, Pfam, EC and PlantTFDB databases, a 69% annotation rate was achieved. Differential expression analysis revealed the regulation of 364 genes in response to wounding. The wound responses in D. cymosum included key mechanisms relating to signalling cascades, phytohormone regulation, transcription factors and defence-related secondary metabolites. However, the role of fluoroacetate in inducible wound responses remains unclear. Bacterial fluorinases were searched against the D. cymosum transcriptome but transcripts with homology were not detected suggesting the presence of a potentially different fluorinating enzyme in plants. Nevertheless, the transcriptome produced in this study significantly increases genetic resources available for D. cymosum and will assist with future research into fluorometabolite-producing plants.

Investment of both essential fatty and amino acids to immunity varies depending on reproductive stage.

Trade-offs among the key life-history traits of reproduction and immunity have been widely documented. However, the currency in use is not well-understood. We investigated how reproducing female side-blotched lizards, Uta stansburiana, allocate lipids versus proteins when given an immune challenge. We tested whether lizards would invest more in reproduction or immunity depending on reproductive stage. Females were given stable isotopes (15 N-leucine and 13 C-1-palmitic acid), maintained on a regular diet and given either a cutaneous biopsy or a sham biopsy (control). Stable isotopes were monitored and analyzed in feces and uric acid, skin biopsies, eggs, and toe clips. We found that lizards deposited both proteins and lipids into their healing wounds (immune-challenged), skin (control), and eggs (all) and that catabolism of proteins exceeded incorporation into tissue during wound-healing. Specifically, we found that healed biopsies of wounded animals had more leucine and palmitic acid than the nonregrown skin biopsies taken from unwounded control animals. Earlier in reproduction, lizards invested relatively more labeled proteins into healing their wound tissue, but not into unwounded skin of control animals. Thus, reproduction is sometimes favored over self-maintenance, but only in later reproductive stages. Finally, we documented positive relationships among the amount of palmitic acid deposited in the eggs, the amount of food eaten, and the amount of palmitic acid excreted, suggesting higher turnover rates of lipids in lizards investing highly in their eggs.

Control of Penicillium postharvest disease of Valencia sweet orange using rosemary extract and thiabendazol

In order to evaluate the effect of rosemary extract and thiabendazol fungicide on postharvest rot of Valencia sweet orange, an experiment was performed as factorial arrangement in a completely randomized design with four replications. The first factor was storage type including cool (4°C) and room temperature (25°C) storages and the second factor was treatment type including unwounded control, wounded control, wounded control inoculated with Penicillium italicum spores and treated with rosemary extract 150, 300 and 450 mg L-1 and thiabendazol 1.0, 1.5 and 2.0 g L-1. The fruits treated with extract and fungicide were inoculated with Penicillium italicum spores. The fruits were stored at room and cool temperature for two months and then the decay percentage, TSS, vitamin C and total acid content were measured. According to the results, the best treatment was thiabendazol 2.0, 1.5 and 1.0 g L-1, respectively, in room temperature storage and rosemary extract could keep fruit quality compared with control.

Regeneration of Defective Epithelial Basement Membrane and Restoration of Corneal Transparency After Photorefractive Keratectomy.

To study regeneration of the normal ultrastructure of the epithelial basement membrane (EBM) in rabbit corneas that had -9.00 D photorefractive keratectomy (PRK) and developed late haze (fibrosis) with restoration of transparency over 1 to 4 months after surgery and in corneas that had incisional wounds. Twenty-four rabbits had one of their eyes included in one of the two procedure groups (-9.00 D PRK or nearly full-thickness incisional wounds), whereas the opposite eyes served as the unwounded control group. All corneas were evaluated with slit-lamp photographs, transmission electron microscopy, and immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin and collagen type III. In the -9.00 D PRK group, corneas at 1 month after surgery had dense corneal haze and no evidence of regenerated EBM ultrastructure. However, by 2 months after surgery small areas of stromal clearing began to appear within the confluent opacity (lacunae), and these corresponded to small islands of normally regenerated EBM detected within a larger area of the excimer laser-ablated zone with no evidence of normal EBM. By 4 months after surgery, the EBM was fully regenerated and the corneal transparency was completely restored in the ablated zone. In the incisional wound group, the two dense, linear corneal opacities were observed at 1 month after surgery and progressively faded by 2 and 3 months after surgery. The EBM ultrastructure was fully regenerated at the site of the incisions, including around epithelial plugs that extended into the stroma, by 1 month after surgery in all eyes. In the rabbit model, spontaneous resolution of corneal fibrosis (haze) after high correction PRK is triggered by regeneration of EBM with normal ultrastructure in the excimer laser-ablated zone. Conversely, incisional wounds heal in rabbit corneas without the development of myofibroblasts because the EBM regenerates normally by 1 month after surgery. [J Refract Surg. 2017:33(5):337-346.].

Expression profiling in Pinus radiata infected with Fusarium circinatum

Fusarium circinatum is the causal agent of pitch canker, a serious disease that affects numerous Pinus species worldwide. Pinus species have varying degrees of susceptibility to this pathogen, being Pinus radiata one of the most susceptible species. Quantitative phenotypic variation and intermediate heritability in response to F. circinatum have been observed by studies of controlled inoculations in several families of P. radiata, suggesting the existence of a genetic component to resistance. Recent experiments in conifer genomics have identified several genes with functions that confer resistance to various biotic and abiotic stresses. In this investigation, high-throughput mRNA sequencing (RNA-seq) is used to identify differentially expressed genes during the interaction of P. radiata with F. circinatum at several time points including uninfected plants at 2, 6, and 12 days post inoculation (dpi) in two contrasting genotypes. The 470,612 Roche 454-HQ reads were de novo assembled into a transcriptome containing 26,215 unigenes with an average length of 1523 bp. Functional annotation indicated that 86% had significant protein alignments and 23,897 (91%) could be assigned to one or more Gene Ontology (GO) terms. Pairwise comparisons of the differentially expressed genes, up and downregulated between the inoculated genotypes, wounded and unwounded control for the resistant genotype at time 2, 6 and 12 dpi were performed. A total of 39,242 and 51 commonly upregulated genes in response to pitch canker infection at 2, 6, and 12 dpi, respectively, during three pairwise comparisons were identified. This RNA-seq analysis identified a total of 293 putative candidate genes and validated a set of genes involved in cellular processes and defense mechanisms, among them, 32 upregulated genes have been associated with defense response in plants against pathogens, thereby providing the first comprehensive evaluation of the putative mechanisms of F. circinatum resistance in pine.

Temporal and spatial expression of terpene synthase genes associated with agarwood formation in Aquilaria malaccensis Lam

The diseased wood, agarwood, from the tropical tree taxa Aquilaria, is famed for its unique fragrance and medicinal values, mainly due to its richness in secondary metabolites such as the sesquiterpenes. The presence of sesquiterpenes in high numbers and amounts correlates with agarwood of high quality. To understand the synthesis of this important compound, we cloned two candidate genes in the terpenoid synthesis pathway from Aquilaria malaccensis Lam., a major agarwood tree species. The genes encoding sesquiterpene synthase (AmSesTPS1) and δ-guaiene synthase (AmGuaiS1) were successfully cloned from callus RNA, using specific primers derived from transcriptomic data, in a reverse transcription PCR reaction. The full-length complementary DNA (cDNA) sequence of AmSesTPS1 was 1632 bp encoding for 544 amino acids, and AmGuaiS1 was 1644 bp encoding for 547 amino acids. Sequence alignment analysis showed that AmSesTPS1 shared between 99 to 100 % identity with sesquiterpene synthase from Aquilaria sinensis (Lour.) Spreng. while AmGuaiS1 shared between 95 to 99 % identity with δ-guaiene synthases from Aquilaria crassna Pierre ex Lecomte and A. sinensis. The genes were functionally characterised in a time course wounding experiment using 3-year-old living trees. Two types of wood samples were collected: (1) from wounded area (S1) and (2) from 5 cm below the wounded area (S2). AmSesTPS1 was highly expressed after 6 h post wounding for both S1 and S2, at a level three- to six-fold higher than that of the unwounded control (0 h), while AmGuaiS1 was induced after just 2 h of wounding (18- and 5.5-fold at S1 and S2, respectively), after which the expression of both genes was lowered. The average normalised expression of both genes at S1 and S2 indicates the genes were also upregulated in the distal area from the wounding site. It can be deduced that wounding triggers these two genes in the sesquiterpene synthesis pathway, which ultimately leads to agarwood formation.

Epithelial basement membrane proteins perlecan and nidogen-2 are up-regulated in stromal cells after epithelial injury in human corneas

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins–important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

A Rat Model for Muscle Regeneration in the Soft Palate

BackgroundChildren with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. Despite successful surgical repositioning of the muscles, optimal function is often not achieved. Scar formation and defective regeneration may hamper the functional recovery of the muscles after cleft palate repair. Therefore, the aim of this study is to investigate the anatomy and histology of the soft palate in rats, and to establish an in vivo model for muscle regeneration after surgical injury.MethodsFourteen adult male Sprague Dawley rats were divided into four groups. Groups 1 (n = 4) and 2 (n = 2) were used to investigate the anatomy and histology of the soft palate, respectively. Group 3 (n = 6) was used for surgical wounding of the soft palate, and group 4 (n = 2) was used as unwounded control group. The wounds (1 mm) were evaluated by (immuno)histochemistry (AZAN staining, Pax7, MyoD, MyoG, MyHC, and ASMA) after 7 days.ResultsThe present study shows that the anatomy and histology of the soft palate muscles of the rat is largely comparable with that in humans. All wounds showed clinical evidence of healing after 7 days. AZAN staining demonstrated extensive collagen deposition in the wound area, and initial regeneration of muscle fibers and salivary glands. Proliferating and differentiating satellite cells were identified in the wound area by antibody staining.ConclusionsThis model is the first, suitable for studying muscle regeneration in the rat soft palate, and allows the development of novel adjuvant strategies to promote muscle regeneration after cleft palate surgery.

Interleukin-1 receptor role in the viability of corneal myofibroblasts

The purpose of this study was to investigate the role of interleukin-1 (IL-1) in modulating myofibroblast viability in mouse corneas with stromal opacity. Twenty-four female B6; 129S1-Il1r1tm1Roml/J homozygous IL-1RI knockout mice and 24 control B6129SF2/J mice were included in this study. Each mouse had opacity-generating irregular phototherapeutic keratectomy (PTK) performed with an excimer laser in one eye. Groups of 8 mice from each group were euthanized at one month, three months and six months after surgery and the eyes cryo-preserved. The contralateral eye served as unwounded control. Immunohistochemistry was performed for α-smooth muscle actin (SMA) in central sections of all corneas. The TUNEL assay for apoptosis was performed on 8 sections of four eyes from each group. No SMA+ cells were detected in the stroma of unwounded control or knockout corneas. SMA+ myofibroblast density was significantly higher (p < 0.001) in the IL-1RI knockout group than in the control group at one month, three and six months after irregular PTK. Mean TUNEL+ stromal cells in the anterior 50 μm of stroma was significantly lower in the IL-1RI knockout group compared to the control group at six months after irregular PTK (p = 0.04). These results corroborate the findings of recent in vitro work that demonstrated an antagonistic effect of TGFβ and IL-1 on myofibroblast viability, and found that IL-1-triggered myofibroblast apoptosis was suppressed by TGFβ. Thus, IL-1 is an important modulator of myofibroblast viability during corneal wound healing.

Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

Role of COX-2 in lymphangiogenesis and restoration of lymphatic flow in secondary lymphedema

The pathophysiology of secondary lymphedema remains poorly understood. To clarify the roles of cyclooxygenase (COX)-2 in enhancement of lymphangiogenesis during secondary lymphedema, we tested a mouse tail model and evaluated the recurrence of lymph flow. To induce lymphedema, a circumferential incision was made in the tail of anesthetized mice to sever the dermal lymphatic vessels. The maximum diameters of the tails were measured weekly. We found that the diameters of the tails around the wounds were markedly increased after surgery, and reached maximum size 2 weeks after wounding in mice without a COX-2 inhibitor, celecoxib (Celecoxib–). Expression of COX-2 in wound granulation tissues was markedly increased 1 week after surgery compared with unwounded naive control mice. In Celecoxib–, recurrence of lymphatic flow in the wound granulation tissues was detected 3 weeks after surgical treatment. In contrast, lymphatic flow was markedly suppressed in mice treated with celecoxib (Celecoxib+). Newly formed lymphatic structures were identified in the granulation tissues formed at wounded lesions in Celecoxib–, whereas those were markedly suppressed in Celecoxib+. Interstitial tissue pressures in the distal areas of the tail wounds were markedly increased in Celecoxib+ with reduced expression of vascular endothelial cell growth factor (VEGF)-C. F4/80-positive cells were accumulated to the wound granulation tissues in Celecoxib–, and the accumulation of these cells was suppressed in Celecoxib+. Prostaglandin E2 (PGE2) upregulated the expressions of VEGF-A and VEGF-C in cultured macrophages, but not human lymphatic microvascular endothelial cells. The present study therefore suggests that lymphangiogenesis, together with recurrence of lymph flow after surgical induction of lymphedema, is upregulated by COX-2 possibly via generation of PGs.

Chaperonin containing T-complex polypeptide (CCT) subunit expression in oral mucosal wounds and fibroblasts

Mucosal wound healing in adults has been reported to feature diminished scar formation compared to healing skin wounds. We sought to determine if the expression pattern of chaperonin containing T-complex polypeptide (CCT) subunits in mucosal wounds and fibroblasts is different from that observed in skin wounds and fibroblasts. We found that CCT-beta is the only subunit message to be reduced in wounded mucosa versus unwounded control, and this reduction was confirmed at the protein level. In contrast, mRNA levels of CCT-zeta, -delta, -eta, and -epsilon were significantly increased in mucosal wounds. The increase in CCT-eta was also confirmed at the protein level. Expression levels of CCT-alpha, -beta, -delta; -epsilon, and -theta mRNAs were significantly increased in adult mucosal fibroblasts in culture compared to skin-derived fibroblasts. Western blot analyses confirmed a modest increase in CCT-beta in adult mucosal fibroblasts relative to skin fibroblasts, but CCT-eta protein was unaffected. These differences may contribute to the reported difference in healing outcomes between these two tissue types.

A comparison of basal and eye-flush tears for the analysis of cat tear proteins

To identify a rapid and effective tear collection method providing sufficient tear volume and total protein content (TPC) for analysis of individual proteins in cats. Domestic adult short-haired cats (12-37 months; 2.7-6.6 kg) were used in the study. Basal tears without stimulation and eye-flush tears after instillation of saline (10 μl) were collected using microcapillary tubes from animal eyes either unwounded control or wounded with 9-mm central epithelial debridement giving four groups with n = 3. Tear comparisons were based on total time and rate for tear collection, TPC using micro bicinchoninic acid (BCA), tear immunoglobulin A (IgA), total matrix-metalloproteinase (MMP)-9 concentration using sandwich enzyme-linked immunosorbent assay (ELISA) and MMP-9 activity. Eye-flush tears were collected significantly faster than basal tears in wounded eyes with higher rates for tear collection in unwounded control and wounded eyes. TPC was significantly lower in eye-flush tears compared to basal tears. The relative proportion of tear IgA normalized to TPC (% IgA of TPC) was not significantly different between basal and eye-flush tears. In unwounded control eyes, MMP-9 was slightly higher in eye-flush than in basal tears; activity of MMP-9 in both tear types was similar. In wounded eyes, eye-flush tears showed highest MMP-9 levels and activity on Day 1, which subsequently decreased to Day 7. MMP-9 activity in basal tears from wounded eyes did not display changes in expression. Eye-flush tears can be collected rapidly providing sufficient tear volume and TPC. This study also indicates that eye-flush tears may be more suitable than basal tears for the analysis of MMPs following corneal wounding.

Corneal myofibroblast generation from bone marrow-derived cells

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA−GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400× field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9× more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant ( p < 0.01). There were significantly more ( p < 0.01) SMA−GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP− cells, although SMA−GFP− cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60–95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.

Insulin-like growth factor-I in wound healing of rat skin

Growth factors play an important role in orchestrating and enabling the cellular responses required for successful wound healing. In the present study, rat surgical incision was used to investigate insulin-like growth factor-I (IGF-I) expression in skin cells as well as its systemic and cutaneous tissue concentrations during acute phase of wound healing. Thirty two animals were sacrificed at days 2, 3, 5 and 9 after surgery. Eight animals were used as control. Tissue expression of IGF-I in both incisional and periincisional skin areas, as well as in skin of control unwounded animals was determined by immunohistochemistry. Serum and tissue concentrations of IGF-I were measured using RIA. Immunohistochemical analysis revealed enhanced IGF-I immunostaining in the incisional area at day 2 post-wounding. Presence of IGF-I immunoreactivity in the epidermis, as well as in dermal fibroblasts and monocytes within perivascular inflammatory infiltrate suggests its local synthesis. Although serum levels of IGF-I were not altered during wound healing, their tissue contents in the incisional area were significantly increased compared with periincisional area at days 2 and 3 after injury, as well as compared with skin content of unwounded control rats in all examined time points. Obtained results support a paracrine role of IGF-I during the acute phase of wound healing by primary intention in the rat.