Antennal and proboscis sensilla characteristics of Paranthrene tabaniformis (Lepidoptera: Sesiidae).

The poplar clearwing moth, Paranthrene tabaniformis (Lepidoptera: Sesiidae) is a serious wood-boring pest of several trees. The ultramorphology of the antennae and proboscis sensilla of adult P. tabaniformis was examined using scanning electron microscope to determine their structures and sex-specific differences. The results showed that the antennae of both sexes are composed of three segments: scape, pedicel and flagellum. Female antennae are clavate while male antennae are pectinate. The number of flagellomeres for females was significantly greater than for males. Seven different types of sensilla were identified on antennae of both males and females: Böhm sensilla, sensilla squamiformia, sensilla trichodea (three subtypes), sensilla chaetica, sensilla coeloconica, and sensilla auricillica (two subtypes), and apical sensors. Three different types of sensilla were found on the proboscis of adult P. tabaniformis: sensilla styloconica, sensilla chaetica, and sensilla basiconica (three subtypes). The sexual dimorphism difference in the number, distributional pattern, the length and the basal width of various sensilla on the antennae and proboscis were determined. This study clarifies the types and sexual dimorphism of the antennal and proboscis sensilla of adult P. tabaniformis and provides useful theoretical foundations for studies on behavioral mechanisms and functions of sensilla of P. tabaniformis. RESEARCH HIGHLIGHTS: Various types of sensilla on the antennae and proboscis of adults Paranthrene tabaniformis were observed. The sexual dimorphism of various sensilla on the antennae and proboscis were determined.

Outcomes of different lines of keratoconus management in a tertiary eye center in north China.

AIM: To evaluate the treatment selections and outcomes of keratoconus and discuss the grading treatment of keratoconus. METHODS: Medical records of 1162 patients (1863 eyes) with keratoconus treated with rigid gas permeable (RGP), corneal collagen crosslinking, and keratoplasty were reviewed. The patients were grouped according to the CLEK Study. The advanced group was further divided into a <60 D group and >60 D group. The best-corrected visual acuity (BCVA) and topographic data before and after treatment were recorded. RESULTS: In the 761 eyes with steep K<52 D, nonsurgical management accounted for 83.4%, while in the 735 eyes with steep K>60 D, surgical management accounted for 90.6%. A total of 618 eyes had improved BCVA at the final follow-up point (>18mo, P<0.001). When steep K was <52 D, the BCVA in the RGP group was better than those with lamellar keratoplasty (LKP; P=0.028). When steep K was >52 D, the BCVA and topographic astigmatism outcomes showed no differences among the treatment groups. When steep K was >60 D, the BCVA in eyes treated with LKP was worse than those with steep K<60 D (P=0.025). The incidence of steep K progression in the RGP group was higher in advanced group (20.0% vs 10.8%, P=0.019). The probability of future keratoplasty in RGP was higher in advanced group (14.8% vs 7.0%, P=0.027). The incidence of steep K progression in the corneal collagen crosslinking (CXL) group was higher in advanced group (32.3% vs 8.5%, P=0.007). Multivariate logistic regression revealed the following related factors for treatment options: steep K [odds ratio (OR)=1.208, 95%CI: 1.052-1.387], TA (OR=1.171, 95%CI: 1.079-1.270), and TCT (OR=0.978, 95%CI: 0.971-0.984). The level of steep K, TA, and TCT all relates to the treatment choices of both keratoplasty and non-keratoplasty, while steep K provided the highest diagnostic accuracy (AUC=0.947, P<0.001). CONCLUSION: Steep K is an important grading treatment indicator. When steep K is <52 D, RGP lenses should be recommended. It is the best time for LKP when the steep K ranges from 52 to 60 D.

Analysis of risk factors of rapid attenuation of graft endothelium in the early stage after penetrating keratoplasty.

This study aimed to analyze the factors of rapid attenuation of graft endothelium in the early stage after penetrating keratoplasty (PKP), with a view to guiding patients with PKP to better long-term outcomes. This study included 226 patients who underwent PKP with follow-up time >1 year at the Qingdao Eye Hospital of Shandong First Medical University from January 2018 to June 2020. Medical records were retrospectively studied, and donor factors, patient factors, and surgical factors were comparatively analyzed to clarify those affecting the rapid decay of graft endothelium after PKP. The median time between excision and death >60 min and patient age >60 years were risk factors for endothelial cell loss (ECL) rate >30% at 1 month postoperatively. However, a higher percentage of patients with donor age ≤60 years and Optisol preservation solution had endothelial cell density (ECD) >2000 cells/mm2 in the graft at postoperative 1 year. A year after the surgery, patients with corneal endothelial decompensation and immune rejection were at risk for ECD < 1000 cells/mm2. The combined operations had a significant effect on the ECL in the early postoperative period. Patients who underwent combined extracapsular cataract extraction or intraocular lens implantation had a significantly higher rate of ECL at postoperative 1 month than other patients, and no significant effect at postoperative 1 year. However, patients without combined operations have a higher probability of maintaining a high level of graft ECD. The graft diameter also affected postoperative ECL. In patients with a larger graft diameter, attenuation of ECD was slower. The ultimate goal of PKP is to maintain graft transparency for extended periods. The use of younger donors, minimizing unnecessary operation in the anterior chamber, and minimizing immune rejection may maintain a greater donor corneal endothelium in the long term.

Two single mutations in carboxylesterase 001C improve fenvalerate hydrolase activity in Helicoverpa armigera.

Carboxylesterases (CarEs) usually play critical roles in the detoxification of toxic chemicals and therefore may be involved in insecticide resistance in agricultural pests. Previous work has shown that CarE 001C from Helicoverpa armigera was able to metabolize the isomers of cypermethrin and fenvalerate. In this study, seven mutants of CarE 001C with single amino acid substitution were produced and expressed in the Escherichia coli. Enzyme kinetic analysis indicated that all seven mutations dramatically reduced enzymatic activities toward the generic substrate α-naphthyl acetate, but in vitro metabolism assay showed that two of the mutations, H423I and R322L, significantly improved hydrolase activities toward fenvalerate, with their recorded specific activities being 3.5 and 5.1 nM·s-1·mg -1 proteins, respectively. Further, thermostability assay showed that the stability of one mutant enzyme was enhanced. This study will help us better understand the potential of CarEs in insecticide detoxification and resistance in H. armigera.

Increased cGAS/STING signaling components in patients with Mooren's ulcer.

AIM: To explore the expression of cGAS/STING signaling components in Mooren's ulcer (MU). METHODS: Samples were obtained from ten MU patients, and eight residual corneal-scleral rings of healthy donor corneas for controls. Human corneal epithelial cells (HCECs) were used to evaluate the effect of cGAS/STING signaling pathway. Immunohistochemistry (IHC) and Western blot were used to examine the expression of cGAS, STING, and phosphorylated interferon regulatory factor 3 (p-IRF3) in MU tissues. The expression of interferon-ß (IFN-ß) and interferon-stimulated genes (ISGs) was quantified by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The protein levels of cGAS and STING in MU samples were significantly elevated when compared with the healthy controls by Western blot and IHC. After stimulation with cGAMP, real-time PCR and ELISA showed a dramatic increase of IFN-ß and ISGs (containing CXCL10, IFIT1, and IL-6) in HCECs. Moreover, HCECs treated with cGAMP was characterized by increased phosphorylation and more nuclear translocation of IRF3. Meanwhile, increased p-IRF3 was observed in MU samples via IHC and Western blot. CONCLUSION: The pronounced expression of cGAS/STING signaling components in the patients with MU and probably contribute to the onset and development of MU.

Midterm outcomes of penetrating keratoplasty following allogeneic cultivated limbal epithelial transplantation in patients with bilateral limbal stem cell deficiency.

AIM: To evaluate the midterm outcomes of penetrating keratoplasty (PK) following allogeneic cultivated limbal epithelial transplantation (CLET) for bilateral total limbal stem cell deficiency (LSCD). METHODS: Ten patients (10 eyes) with bilateral LSCD were enrolled in this prospective noncomparative case series study. Each participant underwent PK approximately 6mo after a CLET. Topical tacrolimus, topical and systemic steroids, and oral ciclosporin were administered postoperatively. Best-corrected visual acuity (BCVA), intraocular pressure (IOP), ocular surface grading scores (OSS), corneal graft epithelial rehabilitation, persistent epithelial defect (PED), immunological rejection, and graft survival rate were assessed. RESULTS: The time interval between PK and allogeneic CLET was 6.90±1.29 (6-10)mo. BCVA improved from 2.46±0.32 logMAR preoperatively to 0.77±0.55 logMAR post-PK (P<0.001). Kaplan-Meier analysis of mean graft survival revealed graft survival rates of 100% at 12 and 24mo and 80.0% at 36mo. PEDs appeared in 5 eyes at different periods post-PK, and graft rejection occurred in 4 eyes. The total OSS decreased from 12.4±4.4 before allogeneic CLET to 1.4±1.51 after PK. CONCLUSION: A sequential therapy design of PK following allogeneic CLET can maintain a stable ocular surface with improved BCVA despite the relatively high graft rejection rate.

Treatment of bilateral granulomatous lobular mastitis during lactation with traditional Chinese medicine: A case report.

BACKGROUND: Granulomatous lobular mastitis (GLM) is a type of benign chronic inflammatory disease that poses therapeutic challenges to healthcare providers. The diagnosis of GLM relies on tissue biopsy, and incorrect treatment may lead to delayed diagnosis, considerable aesthetic damage, and even mastectomy. CASE SUMMARY: We report the case of a 37-year-old Chinese woman who was lactating and had GLM in both breasts. At the time of treatment, the right breast had a mass of approximately 15 cm × 11 cm, which was hard and had poor mobility. Multiple skin ulcerations and pus spills were also observed on the surface of the breast. The left breast had a mass of about 13 cm × 9 cm, which was hard and had poor mobility. CONCLUSION: Herein, we report a case of bilateral GLM in a lactating woman that was successfully treated with traditional Chinese medicine (TCM), without the requirement for surgery or other treatments. Therefore, TCM may have advantages in the nonsurgical treatment of GLM.

Enhanced hydrolysis of ß-cypermethrin caused by deletions in the glycin-rich region of carboxylesterase 001G from Helicoverpa armigera.

BACKGROUND: Carboxylesterase (CarE) is a major class of enzyme involved in the detoxification of toxic xenobiotics in various insect species. Previous work has shown that the carboxylesterase gene CarE001G found in Helicoverpa armigera is more active and can metabolize synthesized pyrethroids, such as ß-cypermethrin, one of the commonly used commercial insecticides for lepidopteran pest control. In addition, CarE001G is very special as it has a very specific glycine-rich region located adjacent to its C-terminal. But whether mutations in this unique sequence can change the biochemistry and function of CarE001G are unknown. RESULTS: In this study, four variants of CarE001G with different deletions in the glycine-rich region were obtained and functionally expressed in Escherichia coli. The recombinant proteins were purified and confirmed by Western blot and mass spectrometry analyses. These mutant enzymes showed high catalytic efficiency toward the model substrate α-naphthyl acetate. Inhibition study showed that ß-cypermethrin had relatively strong inhibition on CarE activities. In vitro metabolism assay showed that the mutant enzymes significantly enhanced their metabolic activities toward ß-cypermethrin with specific activities between 4.0 and 5.6 nmol L-1 min-1 mg-1 protein. Molecular docking analyses consistently demonstrated that deletion mutations in the glycine-rich region may facilitate the anchoring of the ß-cypermethrin molecule in the active binding pocket of the mutant enzymes. CONCLUSION: The data show that deletion mutations can cause qualitative change in the capacity of CarEs in the detoxification of ß-cypermethrin. This indicates that deletion mutations in the glycine-rich region may have the potential to cause synthesized pyrethroid (SP) resistance in H. armigera in the future. © 2020 Society of Chemical Industry.

Comparison of long-term results after manual and femtosecond assisted corneal trephination in deep anterior lamellar keratoplasty for keratoconus.

AIM: To compare long-term postoperative outcomes of manual and femtosecond assisted corneal trephination in deep anterior lamellar keratoplasty (FS-DALK) for keratoconus. METHODS: In the retrospective study, 17 consecutive eyes that underwent vertical side cut incision FS-DALK and 22 eyes that underwent trephine incision DALK were collected over a 2-year period. Main measurements included postoperative uncorrected-visual acuity (UCVA), corrected distance visual acuity (CDVA), refractive sphere and cylinder, manifest refraction spherical equivalent (MRSE), flat and steep corneal keratometry (K1 and K2), endothelial cell density (ECD), and time of epithelium healing and suture removal. RESULTS: Groups were comparable for diagnosis and preoperative visual acuity. Follow-up averaged 23mo (range, 12-36mo). At 12mo, the mean UCVA was better in the manual-DALK group (P=0.039), and the refractive sphere was lower in the FS-DALK group (P=0.040). MRSE between groups differed at 1, 6, and 12mo postoperatively (P=0.047, 0.025, 0.042, respectively). Mean CDVA, cylinder, K1, K2, corneal astigmatism, ECD, and time of epithelium healing were similar between groups. Stability of MRSE, ECD, and K1 returned sooner after FS-DALK. Initial loosened suture removal time was earlier in the manual-DALK group (P=0.042) while complete suture removal time was similar (P=0.122). CONCLUSION: Manual and femtosecond assisted corneal trephination in DALK are options for advanced keratoconus. FS-DALK do not result in improved visual acuity but it is more stable during the follow-up period. FS-DALK in the present form show limited benefit, so surgical design and parameters still need to be optimized and explored.

Functional Characterization of Two Carboxylesterase Genes Involved in Pyrethroid Detoxification in Helicoverpa armigera.

Insect carboxylesterases are major enzymes involved in metabolism of xenobiotics including insecticides. Two carboxylesterase genes, CarE001A and CarE001H, were cloned from the destructive agricultural pest Helicoverpa armigera. Quantitative real-time polymerase chain reaction showed that CarE001A and CarE001H were predominantly expressed in fat body and midgut, respectively; developmental expression analyses found that the expression levels of both CarEs were significantly higher in fifth-instar larvae than in other life stages. Recombinant CarE001A and CarE001H expressed in the Escherichia coli exhibited high enzymatic activity toward α-naphthyl acetate. Inhibition assays showed that organophosphates had strong inhibition on CarEs activity compared to pyrethroids. Metabolism assays indicated that CarE001A and CarE001H were able to metabolize ß-cypermethrin and λ-cyhalothrin. Homology modeling and molecular docking analyses demonstrated that ß-cypermethrin could fit nicely into the active pocket of both carboxylesterases. These results suggested that CarE001A and CarE001H could play important roles in the detoxification of pyrehtroids in H. armigera.

Ocular bacterial infections at a tertiary eye center in China: a 5-year review of pathogen distribution and antibiotic sensitivity.

AIM: To provide statistical evidence for the use of antibiotics in ophthalmology by assessing the distribution and antibiotic sensitivity of bacterial isolates from ocular specimens with suspected microbial infections. METHODS: This study applied a retrospective analysis of 3690 bacterial isolates from ocular specimens, which were obtained from the conjunctiva, cornea, aqueous humor, vitreous body, and other ocular sites of the patients at Shandong Eye Institute in northern China from January 2013 to December 2017. The parameters assessed mainly included the distribution of isolated bacteria and the results of susceptibility tests for antibiotics. In the analysis of antibiotic sensitivities, the bacteria were divided into four groups according to gram staining, and statistical methods were used to compare their antibiotic sensitivities. RESULTS: Among the 3690 isolated bacterial strains, Staphylococcus epidermidis (2007, 54.39%) accounted for the highest proportion. As for the total isolates, their sensitivity rate to gatifloxacin was up to 90.01%, with four types of gram-stained bacteria being all highly sensitive to it, but their sensitivity rate to levofloxacin was only 51.91%. The sensitivity rate of gram-negative bacilli (G-B) to levofloxacin was 83.66%, significantly higher than the other three types of gram-stained bacteria (P<0.05). Gram-positive cocci (G+C, 97.95%) and gram-positive bacilli (G+B, 97.54%) were more sensitive to vancomycin than gram-negative cocci (G-C, 70.59%) and G-B (68.57%; P<0.05). For fusidic acid, the sensitivity rates of G+C (89.83%) and G+B (73.37%) were significantly higher than that of G-B (29.83%; P<0.05). The gram-negative bacteria's sensitivity rate to cefuroxime was as low as 59.25%, but only G-B was less sensitive to cefuroxime (57.28%), while G-C was still highly sensitive (89.29%). The sensitivity rate of gram-positive bacteria to moxifloxacin was as high as 80.28%, but only G+C was highly sensitive to moxifloxacin (81.21%), while G+B was still less sensitive (32.00%). CONCLUSION: Staphylococcus epidermidis is the predominant isolate in all ocular specimens with bacteria. Gatifloxacin is more suitable for topical prophylactic use than levofloxacin in ophthalmology when necessary. Vancomycin and fusidic acid both have better effects on gram-positive bacteria than gram-negative bacteria. More accurate antibiotic sensitivity analysis results can be obtained when a more detailed bacterial classification and more appropriate statistical methods are performed.

Identification and biochemical characterization of carboxylesterase 001G associated with insecticide detoxification in Helicoverpa armigera.

Carboxylesterases (CarEs) are a major class of detoxification enzymes involved in insecticide resistance in various insect species. In this study, a novel CarE 001G was isolated from the cotton bollworm Helicoverpa armigera, one of the most destructive agricultural insect pests. The open reading frame of 001G has 2244 nucleotides and putatively encodes 747 amino acid residues. The deduced CarE possessed the highly conserved catalytic triads(Ser-Glu-His) and pentapeptide motifs (Gly-X-Ser-X-Gly), suggesting 001G is biologically active. The truncated 001G was successfully expressed in Escherichia coli, and the recombinant proteins were purified and tested. The enzyme kinetic assay showed the purified proteins could catalyze two model substrates, α-naphthyl acetate and ß-naphthyl acetate, with a kcat of 8.8 and 2.3 s-1, a Km of 9.6 and 16.2 µM, respectively. The inhibition study with pyrethroid, organophosphate and neonicotinoid insecticides showed different inhibition profile against the purified CarE. The HPLC assay demonstrated that the purified proteins were able to metabolize ß-cypermethrin, λ-cyhalothrin and fenvalerate insecticides, exhibiting respective specific activities of 1.7, 1.4 and 0.5 nM/min/mg protein. However, the purified proteins were not able to metabolize the chlorpyrifos, parathion-methyl, paraoxon-ethyl and imidacloprid. The modeling and docking analyses consistently demonstrated that the pyrethroid molecule fits snugly into the catalytic pocket of the CarE 001G. Collectively, our results suggest that 001G may play a role in pyrethroids detoxification in H. armigera.

Increased succinate receptor GPR91 involved in the pathogenesis of Mooren's ulcer.

AIM: To investigate the expression of succinate receptor GPR91 and its pathogenic roles in Mooren's ulcer (MU). METHODS: Biopsy specimens were obtained from 7 patients with MU and 6 healthy donors. The expression of GPR91 in MU tissues was evaluated using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Succinate was used to activate GPR91 signaling, and the effect of GPR91 on the expression of interleukin-1ß (IL-1ß), NLRP3, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-13 (MMP-13) in human peripheral blood mononuclear cells (PBMCs) was determined. The influence of GPR91 on the nuclear factor-κB (NF-κB) signaling in PBMCs was investigated by detecting the phosphorylation of p65. Moreover, the expression of IL-1ß, VEGF, MMP-13 and phosphorylated p65 (p-p65) in the tissues of MU was examined by qRT-PCR or IHC. RESULTS: GPR91 mRNA expression showed a higher level in the MU group than in the healthy control group. IHC analysis also revealed that the expression of GPR91 was elevated in patients with MU compared with healthy controls. Moreover, ligation of GPR91 with succinate promoted the lipopolysaccharide-induced production of NLRP3, IL-1ß, VEGF and MMP-13 in PBMCs through increased phosphorylation of p65. Pharmacological inhibition of the NF-κB signaling reversed GPR91 induced production of NLRP3, IL-1ß, VEGF and MMP-13. These findings, coupled with the elevated amounts of IL-1ß, VEGF, MMP-13 and p-p65 observed in the MU biopsies, constituted a rational basis for the involvement of GPR91 in the pathogenesis of MU. CONCLUSION: This study indicates the increased succinate receptor GPR91 in conjunctival or corneal tissues is involved in the pathogenesis of MU through elevated NF-κB activity, which may provide a new therapeutic target for MU.

[Preliminary study on the use of DNA suspension array for the identification of corneal pathogenic fungi].

OBJECTIVE: To develop a multiplex, microsphere-based DNA suspension array for the identification of important pathogenic fungi of cornea and to study the feasibility of its application in the clinical diagnosis of fungal keratitis. METHODS: Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Aspergillus fumigatus and Aspergillus flavus, which covered about 80% of pathogenic fungi of fungal keratitis, were chosen as target species of this study. Five species-specific capture probes were designed in the internal transcribed spacer (ITS) regions of ribosomal DNA and synthesized with 5' amino modifier C12 to covalently bind to different sets of fluorescent beads. Biotinylated amplicons of 5 reference strains and 42 clinical strains were generated with a pair of universal primers to yield fragments for detection. Comparison between single species detection and multiplex detection were designed, as well as detection between array and agarose gel electrophoresis (AGE). Spearman rank correlation analysis and t test were applied to evaluate the specificity, sensibility and reproducibility of suspension array. RESULTS: Five reference strains and 40 of 42 (95.2%) clinical strains were correctly identified within 3 h post-PCR amplification while 2 other clinical strains were not identified because of their high background fluorescence intensity. Positive S/B ranged from 5.6 to 13.3. There was no significant difference between respective detection and mixed detection of 5 species (t = 0.2524, P = 0.8132). The sensitivity limit for this assay was determined to be 0.94 ng PCR products. The MFI presented positive correlation with amount of PCR products (r(s) = 1.0000, P < 0.01). Coefficient variation of four repeated detections was 1.8% - 13.7%. CONCLUSION: The suspension array is a rapid, sensitive and specific method for the identification of the most important species of corneal pathogenic fungi and might be used in the clinical laboratories.

Characterization of calreticulin expression in mouse endometrium during embryo implantation.

Calreticulin (CRT), a Ca(2+)-binding storage protein and chaperone in the endoplasmic reticulum, modulates cell adhesiveness and integrin-dependent Ca(2+) signaling. However, the role of CRT during implantation remains poorly understood. In the present study, we characterized the expression of CRT mRNA and the protein in mouse endometria from pregnancy DI to D7. Real-Time PCR and in situ hybridization results showed that the levels of CRT mRNA in the endometria of pregnant mice were significantly higher than those of non-pregnant mice (P<0.05), and increased gradually from pregnancy DI to D4, reaching the máximum level on D4, followed by a plateau from D4 to D7. Using immunofluorescence histochemistry and western blot, changes of CRT expression in the endometria of pregnant mice were consistent with the expression of CRT mRNA. Furthermore, antisense CRT oligodeoxynucleotide was injected into the uterus horns of pregnant mice (D3) to investígate its effect on embryo implantation. The result showed that the number of implanted embryos markedly decreased in the side of uterine horns receiving antisense CRT oligodeoxynucleotide(í(>)<0.05). These findings suggest that CRT may play an important role in embryo implantation in mice.

Characterization of Calreticulin Expression in Mouse Endometrium during Embryo Implantation

Calreticulin (CRT), a Ca2+-binding storage protein and chaperone in the endoplasmic reticulum, modulates cell adhesiveness and integrin-dependent Ca2+ signaling. However, the role of CRT during implantation remains poorly understood. In the present study, we characterized the expression of CRT mRNA and the protein in mouse endometria from pregnancy DI to D7. Real-Time PCR and in situ hybridization results showed that the levels of CRT mRNA in the endometria of pregnant mice were significantly higher than those of non-pregnant mice (P<0.05), and increased gradually from pregnancy DI to D4, reaching the máximum level on D4, followed by a plateau from D4 to D7. Using immunofluorescence histochemistry and western blot, changes of CRT expression in the endometria of pregnant mice were consistent with the expression of CRT mRNA. Furthermore, antisense CRT oligodeoxynucleotide was injected into the uterus horns of pregnant mice (D3) to investígate its effect on embryo implantation. The result showed that the number of implanted embryos markedly decreased in the side of uterine horns receiving antisense CRT oligodeoxynucleotide(í><0.05). These findings suggest that CRT may play an important role in embryo implantation in mice.