Objective To perform single-molecule, real-time sequencing of ceftazidime-avibactam (CAZ-AVI)-resistant Pseudomonas aeruginosa and to investigate the mechanism underlying ceftazidime-avibactam resistance in P. aeruginosa. Methods The susceptibility of 89 P. aeruginosa isolates randomly sampled from clinical specimens in Sanming First Hospital Affiliated to Fujian Medical University from November 2021 through July 2023 to common antimicrobial agents was tested, and the minimum inhibitory concentration (MIC) of CAZ-AVI was determined against P. aeruginosa with a broth microdilution assay, with CAZ-AVI MICs of 8 mg/L and lower defined as susceptible and 16 mg/L and higher as resistant. The expression of drug-resistant genes ampC, oxa-488, oprD, mexA, oxa-10, oxa-14, vim and tem was quantified in P. aeruginosa using a real-time quantitative reverse transcription PCR (qPCR) assay. CAZ-AVI-susceptible and -resistant P. aeruginosa isolates from the same case were selected for PacBio single-molecule, real-time sequencing, and sequencing results were subjected to genome structure and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations. Results The 89 P. aeruginosa isolates showed a relatively high level of resistance to meropenem (75.28%) and imipenem (74.16%) and the highest susceptibility to amikacin (91.01%). There were 49 CAZ-AVI-resistant P. aeruginosa isolates and 40 susceptible isolates. qPCR assay detected lower oprD gene expression in CAZ-AVI-resistant P. aeruginosa isolates [0.104 (2.385)] than in susceptible isolates [0.551 (17.885)] (Z = -2.958, P < 0.01), and there were no significant differences between CAZ-AVI-susceptible and -resistant P. aeruginosa isolates in terms of ampC, oxa-488, mexA or tem gene expression (all P values > 0.05), while oxa-10, oxa-14 and vim gene was expressed in few P. aeruginosa isolates. There were 1 729, 3 936, 3 737 and 3 955 genes in CAZ-AVI-resistant P. aeruginosa isolates PA-762 and PA-M174 and susceptible isolates PA-885 and PA-808 that were annotated to GO terms, with the highest numbers of genes enriched in the molecular function of catalytic activity, high numbers of genes enriched in biological processes of metabolic process, single-organism process and cellular process, and high numbers of genes enriched in cellular components of cell and cell membranes. There were 1 803, 4 084, 3 915 and 4 066 genes in the PA-762, PA-M174, PA-885 and PA-808 isolates enriched in the KEGG signaling pathway, and the majority of genes were enriched in four primary signaling pathways of metabolism, genetic information processing, environmental information processing and cellular process, with the highest number of genes associated with metabolic pathways. Both CAZ-AVI-resistant P. aeruginosa isolates PA-762 and PA-M174 carried multiple efflux pumps systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM. Single nucleotide substitution was found at position 169 in the DNA sequence of the PA-762 isolate, leading to substitution of serine for glycine at position 57 in the protein sequence, and there are deletions of two bases at positions 307 and 308 in the DNA sequence of the PA-M174 isolate, leading to substitution of threonine for arginine at position 103 in the protein sequence. Conclusion Mutation or downregulation of oprD gene may lead to CAZ-AVI resistance in P. aeruginosa.

Objective:To investigate effects of the autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine on viral structures and biosynthesis of functional proteins in dorsal root ganglia in a guinea pig model of varicella-zoster virus (VZV) infection, and to explore their possible mechanisms.Methods:VZV was cultured and proliferated in human embryonic lung fibroblasts (HELFs) , and peripheral blood mononuclear cells (PBMCs) were isolated from guinea pigs. VZV-HELFs and PBMCs were co-cultured for 18-20 hours, and VZV-PBMCs were collected by centrifugation. Thirty-two guinea pigs were randomly and equally divided into 4 groups (8 mice in each group) : blank control group was injected with autologous PBMCs via the medial canthal venous plexus; autophagy inhibition group, autophagy induction group, and VZV infection group were intraperitoneally injected with 3 mg/kg 3-methyladenine solution, 0.5 mg/kg rapamycin solution, and the same volume of 0.9% NaCl solution respectively, followed 2 hours later by injections with 50 μl of VZV-PBMCs via the medial canthal venous plexus. Fourteen days later, the guinea pigs in each group were sacrificed, and dorsal root ganglion tissues were collected. The transmission electron microscope was used to observe the morphology of virus particles, as well as the morphology and number of autophagic vesicles, Western blot analysis was performed to determine the expression of VZV nucleocapsid protein (NCP) , immediate-early protein 62 (IE62) , and autophagy-related proteins Beclin-1 and p62, and immunohistochemical study to determine the expression of anti-VZV antibodies in VZV-infected dorsal root ganglia. Statistical analysis was carried out by using two-independent-sample t test, one-way analysis of variance, least significant difference- t test or Kruskal-Wallis H test. Results:Nucleocapsid-containing virions and scattered autophagosomes were seen in the dorsal root ganglia in the VZV infection group under the transmission electron microscope. The number of autophagic vesicles significantly differed among the blank control group, VZV infection group, autophagy induction group and autophagy inhibition group ( M[ Q1, Q3]: 0, 5[4, 6], 7[5, 9], 0, respectively; H = 135.60, P < 0.01) , and was significantly higher in the VZV infection group than in the blank control group and autophagy inhibition group (both P < 0.05) , as well as in the autophagy induction group than in the autophagy inhibition group ( P<0.05) , but there was no significant difference between the VZV infection group and autophagy induction group ( P>0.05) . Western blot analysis showed that the expression level of IE62 protein was significantly higher in the VZV infection group (1.49 ± 0.06) than in the blank control group (0.50 ± 0.09, t = 9.17, P < 0.05) ; the expression of anti-VZV antibodies was significantly lower in the autophagy inhibition group than in the autophagy induction group and VZV infection group ( t = 9.24, 7.78, respectively, both P < 0.01) , while there was no significant difference between the autophagy induction group and VZV infection group ( P > 0.05) . Conclusion:Autophagy occurred in the dorsal root ganglia of guinea pigs after VZV infection; the inhibition of autophagy could affect the structure of VZV and decrease the expression of VZV functional proteins in the dorsal root ganglia of guinea pigs.

Objective:To investigate the causes of secondary glaucoma after vitrectomy for familial vitreous amyloidosis associated with transthyretin (TTR) gene Gly83Arg mutation.Methods:A retrospective case study. From January 2008 to January 2020, 13 cases (23 eyes) with hereditary vitreous amyloidosis and treated by vitrectomy in the Affiliated Hospital of Zunyi Medical University were collected. Among them, there were 7 males with 12 eyes and 6 females with 11 eyes. The average age was 43.0±4.8 years. All the affected eyes underwent standard three-channel vitrectomy through the flat part of the ciliary body. According to whether complete vitreous detachment (PVD) was formed during the operation, it was divided into complete PVD group and incomplete PVD group; according to the occurrence time of secondary glaucoma and vitreous amyloidosis after surgery, it was divided into 1-12 months group and 13-36 months group, >37 months group. The average follow-up time after surgery was 36.7±6.0 months. The incidence of secondary glaucoma and the recurrence rate of vitreous amyloidosis between groups were compared by χ2 test; the correlation between recurrence of vitreous amyloidosis and secondary glaucoma after surgery was analyzed by Spearman rank correlation analysis. Results:Among the 23 eyes, there were 8 eyes in the complete PVD group and 15 eyes in the incomplete PVD group, respectively. Vitreous amyloidosis recurred in 15 eyes (65.22%, 15/23) after surgery. There were 14 (93.30%, 14/15) and 1 (6.70%, 1/15) eyes in the incomplete PVD group and the complete PVD group, respectively; the comparison of the recurrence rate of vitreous amyloidosis between the two groups was statistically significant ( χ2=11.676, P<0.01). 1-12 months group, 13-36 months group, >37 months group included 1 (4.35%, 1/23), 12 (52.17%, 12/23), 2 (8.70%, 2/23) Only eye. The recurrence rate in the 13-36 months group was significantly higher than that in the 1-12 months group and >37 month group. Secondary glaucoma occurred in 11 eyes (47.80%, 11/23) after surgery. 1-12 months group, 13-36 months group, above 37 months group were 1 (4.35%, 1/23), 8 (34.78%, 8/23), 2 (8.70%, 2/23) eyes. The incidence of secondary glaucoma in the 13-36 months group was higher than that in the 1-12 months group and >37 months group. Among 11 eyes with secondary glaucoma, 10 eyes had recurrence of vitreous amyloidosis after surgery, and 1 eye had no recurrence. The results of Spearman rank correlation analysis showed that there was a positive correlation between the recurrence of vitreous amyloidosis and the occurrence of secondary glaucoma ( rs=0.516, P=0.012). Conclusion:The incidence of secondary glaucoma after vitrectomy in a family with vitreous amyloidosis caused by the Gly83Arg mutation of TTR gene is higher, and its occurrence is significantly positively correlated with the recurrence of vitreous amyloidosis.

Objective To investigate the changes of component and morphology in internal carotid vulnerable plaque,for helping to make clinical intervention strategy individually. Methods A total of 47 patients with internal carotid vulnerable plaques and primary hypertension underwent 2 high-resolution and multi-contrast MRI scans, from March 2008 to April 2014 were retrospectively reviewed. At baseline, the plaque was mainly located at the proximal internal carotid artery,and maximum plaque thickness ≥1.5 mm with intraplaque hemorrhage(IPH)and(or)thin or ruptured fibrous cap.Interscan interval was 0.5 years and above. Patients with carotid occlusion or surgery were excluded. Morphological measurements included maximum plaque thickness, maximum plaque area and cross-sectional vessel area (CSVA) on the level of plaque with maximum thickness. The paired-samples t test was performed to compare the difference of plaque morphology between baseline and follow-up carotid MRI.Results The interscan interval was 1.83 (1.59,1.99)years for 47 internal carotid vulnerable plaques.One case(interscan interval 2.16 years)showed IPH within those 11 plaques without IPH at baseline,and one case(interscan interval 1.42 years)had new incident IPH within those 36 plaques with IPH at baseline. Maximum plaque thickness increased significantly from(3.94±1.44)mm to(4.24±1.68)mm(t=2.30,P<0.05)by 5.14%(-3.83,11.34)% per year. Maximum plaque area increased significantly from(49.19±21.15)mm2to(56.03±24.91)mm2(t=3.87,P<0.01)by 6.67%(-2.26,19.60)% per year.CSVA increased significantly from(66.22±27.51)mm2to(73.68±31.47)mm2(t=4.08,P<0.01)by 5.18%(-1.63,12.34)% per year.Conclusion The progression of component,burden and outer remodeling in the internal carotid vulnerable plaque may be faster in hypertension, therefore reasonable intervention strategy and regular follow-up carotid MRI should be performed.