Effects of switching from intravitreal injection of aflibercept to faricimab on ocular blood flow in patients with diabetic macular edema.

We assessed the short-term effects of switching from intravitreal aflibercept (IVA) to intravitreal faricimab (IVF) on ocular blood flow in patients with treatment-resistant diabetic macular edema (DME). The medical records of 15 patients with DME who had received IVA injection ≥ 3 months before were retrospectively reviewed. The best-corrected visual acuity, central macular thickness (CMT) on optical coherence tomography, and mean blur rate (MBR) of all disc areas on laser speckle flowgraphy were measured before, 1 week after, and 4 weeks after IVA and IVF, respectively. The changes in visual acuity showed no significant difference after switching from IVA to IVF (P = 0.732). The mean CMT decreased significantly during the follow-up period (both P < 0.001). MBR showed no significant difference during the follow-up period (P = 0.26). However, it decreased significantly 4 weeks after IVF (P = 0.01) compared with the baseline value, but not 4 weeks after IVA (P = 0.074). A significant association was observed between decreased MBR and decreased CMT in patients who received IVF (correlation coefficient: 0.501, P = 0.005) but not in those who received IVA (P = 0.735). Thus, IVF maintained ocular blood flow reduction, although no significant differences in visual acuity and CMT changes were observed compared to IVA.

Impairment of flicker-induced increase in retinal blood flow in diabetic pigs.

PURPOSE: To evaluate retinal blood flow (RBF) regulation in response to RBF stress in maturity-onset diabetes of the young type 3 (MODY3) pigs. STUDY DESIGN: Case-control study. METHODS: MODY3 pigs (diabetes mellitus [DM] group, n = 8) transfected with the human mutant hepatocyte nuclear factor-1⍺ and normal pigs of the same age (normal group, n = 8) were used as subjects. After confirming DM onset, the experiment was performed under inhalation anesthesia with isoflurane at 2 months of age before the cataract progressed. Ocular blood flow was assessed by calculating the optic papillary mean blur rate using laser speckle flowgraphy, modified for pig eye measurements. After baseline ocular blood flow measurements, flicker stimulation (12 Hz, 3 min) was applied, and ocular blood flow was measured over time. RESULTS: Blood glucose was 81.8 ± 5.1 mg/dL in the normal group and 311.4 ± 23.1 mg/dL in the DM group (mean ± standard error). The percent change in ocular blood flow at 3 min after flicker stimulation was +31.0 ± 10.9% in the normal group and -6.6 ± 6.5% in the DM group compared to the preload value, and the difference was statistically significant (Mann-Whitney test, P = 0.015). CONCLUSION: RBF response to flicker stimulation is reduced at 2 months of age in MODY3 pigs, suggesting that retinal neurovascular coupling is impaired from the early onset of DM.

Population Analysis of Daptomycin-non-Susceptible Methicillin-Resistant Staphylococcus aureus Reveals the Presence of Variants That Contribute to Daptomycin Resistance.

In this study, population analysis (PA) of methicillin-resistant Staphylococcus aureus (MRSA), before and after long-duration daptomycin (DAP) treatment, was used to detect subpopulations with different susceptibilities to DAP and to verify the changes in the number of resistant cells. Furthermore, we aimed to characterize the bacteriology of the variants present in the non-susceptible cell subpopulation. A DAP non-susceptible (NS) MRSA phenotype (D2) that emerged from a DAP- susceptible MRSA phenotype (D1) during treatment of an open wound, was used for testing. We performed bacteriological and genetic analyses of cryptic DAP-NS MRSA variants detected by PA to study the variants present in the resistant cell subpopulation. PA results suggest that MRSA adapted to survival in the presence of DAP are selected leading to reduced susceptibility. Within the cell population growing in media containing 2.0 mg/L of DAP, three variants with different pigment production and colony size were detected. Variant 3 was an orange colony due to enhanced production of staphyloxanthin. Our results revealed that the DAP minimum inhibitory concentration (MIC) value increased two-fold (4 mg/L) in variant 3, in which pigment production was most enhanced, compared to the parental strain D2. In conclusion, our results indicate that long-duration DAP treatment can lead to the emergence and increased proportion of DAP-NS subpopulations. Furthermore, slow-growing variants that can be detected only under antimicrobial selective pressure are present among DAP-NS cells, suggesting that these variants may also contribute to the development of DAP resistance.

Epigenetic effects of ceftriaxone-resistant Neisseria gonorrhoeae FC428 mosaic-like sequences found in PenA sequences unique to Neisseria subflava and related species.

OBJECTIVES: The aim of this study was to explore the origin of the PenA mosaic amino acid sequence in the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone. METHODS: The penA sequences of 27 Neisseria subflava pharyngeal isolates were determined by the Sanger method and penA sequences of 52 isolates from nine Neisseria species were obtained from the NCBI database. Comparative analysis of each PenA sequence was performed by multiple sequence alignment using ClustalW. In vitro resistance acquisition experiments were conducted to investigate the possibility of selection pressure by cefixime-induced amino acid substitution mutations in PenA. RESULTS: All N. subflava strains, including two with low susceptibility to expanded-spectrum cephalosporins (ESCs), possessed the majority of the PenA FC428 sequence. Furthermore, a number of strains, but not all, of closely related species of N. subflava showed similar results. PenA FC428 sequences were also found in some strains of distantly related species. No new mutations in the penA sequence were observed in colonies with increased MIC in in vitro resistance acquisition experiments. CONCLUSIONS: This study provides strong evidence that the FC428 PenA mosaic sequence originated from N. subflava and related species among oral commensal Neisseria species. The results of in vitro resistance acquisition experiments also suggested that one of the PenA FC428-like sequence gene polymorphisms resulted in the expression of ESC resistance. Furthermore, many of the PenA FC428 mosaic sequences were thought to be involved in the so-called epistasis effect that regulates the expression of resistance, without directly contributing to the resistance level itself.

Dilation of porcine retinal arterioles to nobiletin, a polymethoxyflavonoid: Roles of nitric oxide and voltage-dependent potassium channel.

We examined the effects of nobiletin, a polymethoxyflavonoid, on the retinal microvascular diameter to determine if they depend on the endothelium and/or smooth muscle to reveal the signaling mechanisms involved in this vasomotor activity. Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded diametric responses to nobiletin. The retinal arterioles dilated in a nobiletin concentration-dependent (100 pM-10 µM) manner and decreased by 50% after endothelial removal. The nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME), reduced nobiletin-induced vasodilation comparable to denudation. Blockade of soluble guanylyl cyclase by 1H-[1,2,4] oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) produced a similar inhibitory effect as that by L-NAME. Nobiletin-induced vasodilation was also inhibited by the nonselective potassium channel inhibitor, tetraethylammonium (TEA), and the voltage-gated K (Kv) inhibitor, 4-aminopyridine. Co-administration of L-NAME and TEA almost eliminated nobiletin-induced vasodilation. Nobiletin elicits both endothelium-dependent and -independent dilation of retinal arterioles mediated by NO release and Kv channel activation, respectively.

Universally Exhaustive Generation of Molecular Structures and Prediction of Their Electronic States Using Machine Learning for N-type Organic Transistor Materials.

We have proposed a new method for the exploration of organic functional molecules, using an exhaustive molecular generator combined without combinatorial explosion and electronic state predicted by machine learning and adapted for developing n-type organic semiconductor molecules for field-effect transistors. Our method first enumerates skeletal structures as much as possible and next generates fused ring structures using substitution operations for atomic nodes and bond edges. We have succeeded in generating more than 4.8 million molecules. We calculated the electron affinity (EA) of about 51 thousand molecules with DFT calculation and trained the graph neural networks to estimate EA values of generated molecules. Finally, we obtained the 727 thousand molecules as candidates that satisfy EA values over 3 eV. The number of these possible candidate molecules is far beyond what we have been able to propose based on our knowledge and experience in synthetic chemistry, indicating a wide diversity of organic molecules.

A pilot study of diabetic retinopathy in a porcine model of maturity onset diabetes of the young type 3 (MODY3).

Diabetic retinopathy (DR) is a leading cause of blindness in the working population. Because novel therapeutic intervention require testing, there is an urgent need for reliable animal models that faithfully replicate DR. Pig eyes have many similarities to human eyes anatomically and physiologically. Thus, attempts have been made to establish porcine models of DR by surgical, pharmaceutical or genetical induction of insulin deficiency, and dietary intervention. A previous study reported a transgenic pig model of maturity onset diabetes of the young type 3 (MODY3) developed signs of severe DR such as hemorrhage and proliferative tissue at the surface of the retina. However, the course of development of DR has not been studied in detail in this model. The purpose of this study was to investigate the early phase of DR in a MODY3. MODY3 and wild-type (WT) pigs underwent fundus photography and fluorescein angiogram (FA) before they developed cataracts. Animals were euthanized at age 1, 4, 7, and 10 months. Whole-mount retina and 10-µm thick paraffinized sections were stained with isolectin B4, and vessel density was determined by MATLAB software. At 4 and 7 months, retinal arterioles were immediately cannulated, and vasomotor action was measured by incubation with bradykinin and sodium nitroprusside. In the MODY3 pigs, fasting blood sugar levels gradually increased up to 500 mg/dL. Vascular tortuosity and yellowish spindle-shaped lesions were confirmed in MODY3 pigs at the age of 7 months; however, no microaneurysms were detected on FA. Compared with age-matched WT pigs, MODY3 pigs showed a significant decrease in blood vessel density in the intermediate and deep vascular plexus at 4 and 7 months of age and a slight decrease in capillary density in the superficial vascular plexus at 7 months of age. In MODY3 pigs, electron microscopy revealed thickening of the capillary basement membrane and leukostasis in the major blood vessels at 10 months of age. Bradykinin-induced dilation of retinal arterioles was diminished in MODY3 pigs as early as 7 months of age. Within 1 year after birth, MODY3 pigs show all typical early vascular lesions of diabetes except for microaneurysm formation. This pilot study suggests that the MODY3 pigs may serve as a suitable DR model to test effects of newly developed compounds on DR.

The emergence of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone by transfer of resistance from an oral Neisseria subflava reservoir of resistance.

BACKGROUND: The ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone was first discovered in Japan in 2015. OBJECTIVES: We investigated the possibility of horizontal gene transfer from Neisseria subflava harbouring the mosaic-like PBP-2 in the emergence of the FC428 clone. We also analysed whether there were fitness costs associated with the sustained international dissemination of the clone. METHODS: Sequencing of the penA gene in ceftriaxone-resistant N. subflava strains was performed. For transformation experiments between donor N. subflava and ciprofloxacin-resistant wild-type penA N. gonorrhoeae recipient, the full-length PCR amplification product of the penA gene, including DUS regions, was used as the donor DNA. Biological fitness of the transformants was measured by growth competition assays. The impact of QRDR and mtrR mutations, which have been reported as compensatory mutations, on fitness was also assessed. RESULTS: The penA mosaic allele of the FC428 clone showed 100%, 91.8%, and 89.8% homology, respectively, with penA genes of three ceftriaxone-resistant N. subflava strains, No. 30, No. 9 and No. 14. Results were consistent with homologous recombination with the donated penA mosaic allele. In co-cultures with the parent strain, transformants showed comparable growth indicating that a gyrA mutation compensates for the fitness cost of mosaic penA alleles. CONCLUSIONS: Our findings support the hypothesis that the FC428 clone was generated by transformation of the mosaic penA allele from oropharyngeal N. subflava to N. gonorrhoeae. Furthermore, it suggests that mutations in the gyrA QRDR region compensate for fitness costs and contribute to the continued transmission of the FC428 clone.

Molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996-2018.

OBJECTIVES: The objective of this study was to investigate the molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Fukuoka, Japan, from 1996-2018. METHODS: Antimicrobial susceptibility to seven antibiotics was determined by the agar dilution method. Molecular characteristics were determined by Sanger sequencing of the blaTEM allele, plasmid typing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, full sequences of the penA gene, encoding penicillin-binding protein 2 (PBP2), of PPNG isolates with reduced susceptibility to cefixime were analysed. RESULTS: Among 50 PPNG isolates, 17 and 33 were collected during 1996-2006 and 2007-2018, respectively. In 1996-2006, blaTEM-1 in African plasmid was most frequent (64.7%), followed by blaTEM-1 in Asian plasmid (29.4%) and blaTEM-135 in Toronto/Rio plasmid (5.9%). In 2007-2018, blaTEM-135 in Toronto/Rio plasmid was predominant (54.5%), followed by blaTEM-1 in African plasmid (36.4%) and blaTEM-135 in Asian plasmid (6.1%). Among isolates with the blaTEM-135-carrying Toronto/Rio plasmid in 2007-2018, a novel genogroup G15576 was predominant (66.7%). Isolates with the TEM-135 ß-lactamase were more resistant to ciprofloxacin but were more susceptible to ceftriaxone and tetracycline than isolates with TEM-1. Seven PPNG isolates less susceptible to cefixime possessed the plasmidic blaTEM-1 allele and had mosaic or non-mosaic alterations within PBP2. CONCLUSION: The proportion of PPNG with the blaTEM135-carrying Toronto/Rio plasmid increased during the last 12 years. The increase in PPNG carrying the blaTEM-135 allele is of particular concern as it is considered a possible direct precursor of an extended-spectrum ß-lactamase (ESBL).

Investigation of the susceptibility trends in Japan to fluoroquinolones and other antimicrobial agents in a nationwide collection of clinical isolates: A longitudinal analysis from 1994 to 2016.

The susceptibilities of clinical isolates to fluoroquinolones and other antimicrobial agents were surveyed to obtain an accurate understanding of trends in incidence and antimicrobial resistance. The samples were collected from across Japan, biennially or triennially, between 1994 and 2016 and a defined level of resistance to fluoroquinolone was determined. Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae exhibited stable and high rates of susceptibility to fluoroquinolones over the period examined. For methicillin-resistant Staphylococcus aureus the rate of resistance to levofloxacin and ciprofloxacin was 81.3-93.5% and 83.2-94.2%, respectively, which was markedly higher than that of methicillin-susceptible S. aureus, while sitafloxacin-resistant methicillin-susceptible and methicillin-resistant S. aureus were isolated at 0.3-0.7% and 16.9-36.5%, respectively. The rate of levofloxacin or ciprofloxacin-resistant Escherichia coli increased from around 2-3% between 1994 and 1998 to around 35% in 2016, but the rate of fluoroquinolone-susceptible Klebsiella pneumoniae stayed high at over 94.6% during the study period. Although no fluoroquinolone-resistance in clinical isolates of Salmonella spp. was detected from 1994 to 2002, the resistance rate increased slightly after 2004 and reached to 1.9%-4.7% in 2016. The rate of fluoroquinolone-susceptible Pseudomonas aeruginosa isolated from urinary tract and respiratory tract infections improved during the period examined from 41.8-67.0% to 91.2-94.2%, and from 78.9-88.5% to 90.1-94.6%, respectively. Against Acinetobacter spp., the susceptibility rate of fluoroquinolones was almost constant at around 90%, but one multidrug-resistant isolate was detected in 2013. Overall, the susceptibility to fluoroquinolones was maintained over 20 years against tested bacteria except for MRSA and E. coli.