Anti-biofilm potential of human senescence marker protein 30 against Mycobacterium smegmatis.

Human senescence marker protein 30 (huSMP30) has been characterized as a multifaceted protein consisting of various enzymatic and cellular functions. It catalyzes the interconversion of L-gulonate and L-gulono-γ-lactone in the ascorbate biosynthesis pathway. Therefore, we hypothesized that it could be a potential anti-biofilm agent against pathogenic bacteria due to its lactonase activity. In order to corroborate this, the huSMP30 was recombinantly expressed, purified, and analyzed for its ability to inhibit Mycobacterium smegmatis biofilm formation, which showed a concentration-dependent inhibition as compared to the untreated control group. Further, in silico analysis was performed to redesign the huSMP30 with enhanced lactonase activity. Molecular docking analysis of the huSMP30 and lactone substrates facilitated the selection of three single amino acid substitutions (E18H, N154Q, and D204V), which were created using a PCR-based site-directed mutagenesis reaction. These mutant proteins and the wild-type huSMP30 were purified, and the effects on the enzymatic activity and biofilm formation were studied. The mutants E18H and D204V showed non-significant effects on specific lactonase activity, catalytic efficiency, and anti-biofilm property; however, the mutant N154Q showed significant improvement in the specific lactonase activity, catalytic efficiency, and inhibition in the biofilm formation. The protein stability analysis revealed that the wild-type huSMP30 and its designed mutants were stable at 37 °C for up to 4 days. In conclusion, the anti-biofilm property of the huSMP30 has been established, and an engineered version, N154Q, inhibits biofilm formation with greater efficiency. Human SMP30 is a versatile protein with multiple cellular and enzymatic functions, however, its anti-biofilm potential has not been explored. Our work presents the method to produce soluble and active huSMP30 in the E. coli expression system and establishes its role as an anti-biofilm agent against Mycobacterium smegmatis owing to its lactonase activity. Our results provide support for the future advancement of huSMP30 as a potential anti-biofilm agent targeting pathogenic Mycobacterium species.

Sequential episodes of perioperative ischemic optic neuropathy after hip surgery.

Perioperative ischemic optic neuropathy (ION) after nonocular surgery is a rare complication leading to permanent and often severe vision loss. Due in part to the low prevalence of this complication, there remains no reliable way to predict which patients will develop ION. We present a patient with sequential episodes of unilateral perioperative ION, both occurring after otherwise uncomplicated hip operations. Patients and physicians should be aware that perioperative ION after one surgery may increase the risk of ION after subsequent surgeries.

Evaluation of the Recombinant Bacterial Chitinases as Anti-proliferative and Anti-migratory Agents for the Human Breast Cancer Cell Line, MCF-7.

Chitinases, a glycosyl hydrolase family 18 members, have a wide distribution in both prokaryotes and eukaryotes, including humans. Regardless of the absence of endogenous chitin polymer, various chitinases and chitinase-like proteins (CLPs) have been reported in mammals. However, several other carbohydrate polymers, such as hyaluronic acid and heparan sulfate, show structural similarities with chitin, which could be a potential target of chitinase and CLPs. Heparan sulfate is part of the integral membrane proteins and involves in cell adherence and migration. Hence, to demonstrate the effect of chitinase on cancer cell progression, we selected two chitinases from Serratia marcescens, ChiB and ChiC, which function as exo- and endo-chitinase, respectively. The ChiB and ChiC proteins were produced recombinantly by cloning chiB and chiC genes from Serratia marcescens. The cell viability of the Michigan Cancer Foundation-7 (MCF-7) cells was studied using different concentrations of the purified recombinant proteins. Cell viability assay was performed using 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide and water-soluble tetrazolium salt, and the effect of ChiB and ChiC on cell proliferation was studied by clonogenic assay. The cell migration study was analysed by wound healing, transwell migration, and invasion assays. Cell cycle analysis of propidium iodide-stained cells and cell proliferation markers such as pERK1/2, pAKT, and SMP30 were also done. It was observed that both ChiB and ChiC were able to impede cell viability, cell migration, and invasion significantly. These observations and our in silico molecular docking analysis suggest that ChiC is a potential anticancer agent and is more efficient than ChiB. Since the ChiC is able to inhibit both cancer cell proliferation and migration, it could be a potential candidate for the treatment of metastatic cancer.

Evaluation of moxifloxacin 0.5% in treatment of nonperforated bacterial corneal ulcers: a randomized controlled trial.

PURPOSE: To compare the equivalence of moxifloxacin 0.5% with a combination of fortified cefazolin sodium 5% and tobramycin sulfate 1.3% eye drops in the treatment of moderate bacterial corneal ulcers. DESIGN: Randomized, controlled, equivalence clinical trial. PARTICIPANTS AND CONTROLS: Microbiologically proven cases of bacterial corneal ulcers were enrolled in the study and were allocated randomly to 1 of the 2 treatment groups. INTERVENTION: Group A was given combination therapy (fortified cefazolin sodium 5% and tobramycin sulfate) and group B was given monotherapy (moxifloxacin 0.5%). MAIN OUTCOME MEASURES: The primary outcome variable for the study was percentage of the ulcers healed at 3 months. The secondary outcome variables were best-corrected visual acuity and resolution of infiltrates. RESULTS: Of a total of 224 patients with bacterial keratitis, 114 patients were randomized to group A, whereas 110 patients were randomized to group B. The mean ± standard deviation ulcer size in groups A and B were 4.2 ± 2 and 4.41 ± 1.5 mm, respectively. The prevalence of coagulase-negative Staphylococcus (40.9% in group A and 48.2% in group B) was similar in both the study groups. A complete resolution of keratitis and healing of ulcers occurred in 90 patients (81.8%) in group A and 88 patients (81.4%) in group B at 3 months. The observed percentage of healing at 3 months was less than the equivalence margin of 20%. Worsening of ulcer was seen in 18.2% cases in group A and in 18.5% cases in group B. Mean time to epithelialization was similar, and there was no significant difference in the 2 groups (P = 0.065). No serious events attributable to therapy were reported. CONCLUSIONS: Corneal healing using 0.5% moxifloxacin monotherapy is equivalent to that of combination therapy using fortified cefazolin and tobramycin in the treatment of moderate bacterial corneal ulcers. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Rate of Falls, Fear of Falling, and Avoidance of Activities At-Risk for Falls in Older Adults With Glaucoma.

PURPOSE: To determine the relationship between glaucoma severity and rate of falls, fear of falling, and avoidance of activities at-risk for falls. DESIGN: Cross-sectional study. METHODS: Patients with glaucoma (n = 138) 55 to 90 years of age with mild (n = 61), moderate (n = 54), or advanced (n = 23) glaucoma in the better eye based on the Glaucoma Staging System and age-matched control subjects (n = 50) were recruited from the Eye Clinics at Washington University, St. Louis, MO. Participants completed questionnaires regarding falls, the fear of falling, and the avoidance of activities at-risk for falls. RESULTS: Of the glaucoma participants, 36% reported ≥1 fall in the previous 12 months compared with 20% of control subjects (adjusted odds ratio [OR] 2.7 [95% confidence interval {CI} 1.18-6.17]; P = .018). Compared with control subjects, the mild glaucoma group trended toward a higher fall risk (adjusted OR 2.43 [95% CI 0.97-6.08]; P = .059) and the advanced group had the highest fall risk (adjusted OR 7.97 [95% CI 2.44-26.07]; P = .001). A greater risk of a high fear of falling and high avoidance of at-risk activities occurred at the moderate stage of glaucoma compared with control subjects (adjusted OR 4.66 [95% CI 1.24-17.49]; P = .023 and adjusted OR 4.49 [95% CI 1.34-15.05]; P = .015, respectively). CONCLUSIONS: Patient education, interventions, and appropriate referrals to minimize falls should be considered in older adults with early glaucoma and continue with advancing disease. Minimizing a patient's fall risk may decrease their fear of falling and avoidance of at-risk activities. Reducing falls, the fear of falling, and the avoidance of at-risk activities may lower morbidity and mortality and improve emotional and social well-being of patients with glaucoma. Am J Ophthalmol 2021;221:•••-•••. © 2021 Elsevier Inc. All rights reserved.

Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork.

PURPOSE: To determine whether activity of carbohydrate metabolism enzymes (aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls. METHODS: Tissue specimens were obtained from trabeculectomy (n=45 open angle glaucoma; Caucasian, average age 61+/-8 years of age of both genders) and from cadaver eyes (n=15 control and n=5 glaucoma; Caucasian, average age 63+/-4 years of both genders). Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1% genapol. Aldolase activity was measured spectrophotometrically at 240 nm absorbance using reaction of 3-phosphoglycerate with hydrazine to form hydrazone. Pyruvate kinase activity was measured by coupling lactate dehydrogenase with NADPH and pyruvate absorbance was measured at 340 nm. Isocitrate dehydrogenase activity was measured using reduction of NADP to NADPH at the characteristic absorbance at 340 nm. Malate dehydrogenase catalyzes the interconversion of L-malate and oxaloacetate using NADP as a coenzyme, quantified by its absorbance at 340 nm. RESULTS: Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively. SDS-PAGE analysis suggests the presence of 4-hydorxynonenal (HNE) modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls. CONCLUSIONS: Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein. Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.

Evaluation of intrastromal injection of voriconazole as a therapeutic adjunctive for the management of deep recalcitrant fungal keratitis.

PURPOSE: To evaluate the role of intrastromal injection of voriconazole in the management of deep recalcitrant fungal keratitis. DESIGN: Interventional case series. SETTING: Cornea services at a tertiary care teaching hospital. PATIENTS: Three eyes of three patients with deep stromal recalcitrant fungal keratitis not responding to topical antifungal medications. Intervention Procedure: Voriconazole 50 micrograms/0.1 ml was injected circumferentially around the fungal abscess in the corneal stroma as an adjunctive to the topical antifungal therapy. MAIN OUTCOME MEASURE: Main outcome measure was a reduction in size of the abscess and resolution of the infection. RESULTS: Before the intracorneal injection, all three eyes had gradually worsening lesions on topical medications. After the intervention, a faster reduction in the size of corneal infiltration was documented and a complete resolution of the ulcers was seen within three weeks in all cases. CONCLUSION: Targeted delivery of voriconazole by intracorneal injection may be a safe and effective way to treat cases of deep-seated recalcitrant fungal keratitis responding poorly to conventional treatment modalities.

Cochlin, intraocular pressure regulation and mechanosensing.

Fluid shear modulates many biological properties. How shear mechanosensing occurs in the extracellular matrix (ECM) and is transduced into cytoskeletal change remains unknown. Cochlin is an ECM protein of unknown function. Our investigation using a comprehensive spectrum of cutting-edge techniques has resulted in following major findings: (1) over-expression and down-regulation of cochlin increase and decrease intraocular pressure (IOP), respectively. The overexpression was achieved in DBA/2J-Gpnmb(+)/SjJ using lentiviral vectors, down-regulation was achieved in glaucomatous DBA/2J mice using targeted disruption (cochlin-null mice) and also using lentiviral vector mediated shRNA against cochlin coding region; (2) reintroduction of cochlin in cochlin-null mice increases IOP; (3) injection of exogenous cochlin also increased IOP; (4) increasing perfusion rates increased cochlin multimerization, which reduced the rate of cochlin proteolysis by trypsin and proteinase K; The cochlin multimerization in response to shear stress suggests its potential mechanosensing. Taken together with previous studies, we show cochlin is involved in regulation of intraocular pressure in DBA/2J potentially through mechanosensing of the shear stress.

Cochlin induced TREK-1 co-expression and annexin A2 secretion: role in trabecular meshwork cell elongation and motility.

Fluid flow through large interstitial spaces is sensed at the cellular level, and mechanistic responses to flow changes enables expansion or contraction of the cells modulating the surrounding area and brings about changes in fluid flow. In the anterior eye chamber, aqueous humor, a clear fluid, flows through trabecular meshwork (TM), a filter like region. Cochlin, a secreted protein in the extracellular matrix, was identified in the TM of glaucomatous patients but not controls by mass spectrometry. Cochlin undergoes shear induced multimerization and plays a role in mechanosensing of fluid shear. Cytoskeletal changes in response to mechanosensing in the ECM by cochlin will necessitate transduction of mechanosensing. TREK-1, a stretch activated outward rectifying potassium channel protein known to act as mechanotransducer was found to be expressed in TM. Cochlin expression results in co-expression of TREK-1 and filopodia formation. Prolonged cochlin expression results in expression and subsequent secretion of annexin A2, a protein known to play a role in cytoskeletal remodeling. Cochlin interacts with TREK-1 and annexin A2. Cochlin-TREK-1 interaction has functional consequences and results in changes in cell shape and motility. Annexin A2 expression and secretion follows cochlin-TREK-1 syn-expression and correlates with cell elongation. Thus cytoskeleton changes in response to fluid shear sensed by cochlin are further mediated by TREK-1 and annexin A2.

Evaluation of umbilical cord serum therapy in acute ocular chemical burns.

PURPOSE: To evaluate the role of umbilical cord serum therapy in cases of acute ocular chemical burns. METHODS: In a double-blind prospective randomized controlled clinical study, 33 eyes of 32 patients with acute ocular chemical burns of grade III, IV, and V severity were randomized into three groups: umbilical cord serum (n = 12), autologous serum (n = 11), and artificial tears (0.5% HPMC+0.3% glycerin; n = 10). In addition, all eyes received standard medical therapy. The parameters evaluated were pain score, size, and area of epithelial defect, extent of limbal ischemia, corneal clarity, and symblepharon formation. The patients were followed up at day 1, 3, 7, 14, and 21 and at the end of months 1, 2, and 3. RESULTS: Mean time to complete epithelialization was 21.16 ± 26.81, 56.6 ± 35.5, and 40.13 ± 35.79 days in cord serum, autologous serum, and artificial tears groups respectively (P = 0.02). By day 21, the mean percentage decrease in epithelial defect diameter was 94.63 ± 11.99 with cord serum compared with 53.17 ± 34.81 and 64.22 ± 42.43 with autologous serum and artificial tears, respectively (P = 0.01). By month 3, the extent of limbal ischemia with cord serum showed a mean percentage decrease of 73.43 ± 25.51 compared with 35.64 ± 25.60 and 43.71 ± 28.71 with autologous serum and artificial tears, respectively (P = 0.008). More patients had clear corneas with cord serum compared with autologous serum and artificial tears (P = 0.048). No significant difference was seen between the groups with regard to symblepharon formation (P = 0.07). CONCLUSIONS: Umbilical cord serum therapy is more effective than autologous serum eye drops or artificial tears in ocular surface restoration after acute chemical injuries. (www.controlled-trials.com number, ISRCTN08131903.).

Aqueous humor dynamics: a review.

Glaucoma is a family of optic neuropathies which cause irreversible but potentially preventable vision loss. Vision loss in most forms of glaucoma is related to elevated IOP with subsequent injury to the optic nerve. Secretion of aqueous humor and regulation of its outflow are physiologically important processes for maintaining IOP in the normal range. Thus, understanding the complex mechanisms that regulate aqueous humor circulation is essential for management of glaucoma. The two main structures related to aqueous humor dynamics are the ciliary body and the trabecular meshwork (TM). Three mechanisms are involved in aqueous humor formation: diffusion, ultrafiltration and active secretion. Active secretion is the major contributor to aqueous humor formation. The aqueous humor flow in humans follows a circadian rhythm, being higher in the morning than at night. The aqueous humor leaves the eye by passive flow via two pathways - the trabecular meshwork and the uveoscleral pathway. In humans, 75% of the resistance to aqueous humor outflow is localized within the TM with the juxtacanalicular portion of the TM being the main site of outflow resistance. Glycosaminoglycan deposition in the TM extracellular matrix (ECM) has been suggested to be responsible for increased outflow resistance at this specific site whereas others have suggested deposition of proteins, such as cochlin, obstruct the aqueous humor outflow through the TM. The uveoscleral outflow pathway is relatively independent of the intraocular pressure and the proportion of aqueous humor exiting the eye via the uveoscleral pathway decreases with age.