Healing responses at the angle after micro-invasive glaucoma surgery-an AS-OCT study.

PURPOSE: To evaluate structural alterations and healing responses in the trabecular meshwork region with optical coherence tomography (AS-OCT) following after gonioscopy assisted transluminal trabeculotomy (GATT) and microincisional trabeculectomy (MIT). METHODS: 73 eyes of 67 patients (M:F = 45:22) with ≥6 months of follow-up after MIT (n = 41) or GATT (n = 32) with or without combined cataract surgery were included for this prospective study. The angle as seen on AS-OCT at 1, 3, 6 months after surgery were evaluated for structural alterations like peripheral anterior synechiae (PAS), hyphema, and hyperreflective scarring responses. The scarring was graded according to the linear extent measured from the centre of the trabecular meshwork (TM) gutter to the sclera/cornea as mild (<250µ), moderate (250-500µ), and severe(˃500µ), while the pattern of scarring was graded as open saucer/gutter, closed gutter, and trench pattern. The association of the need for medication or surgical outcome and clinical variables and AS-OCT parameters including the pattern and severity of scarring were analysed using multivariate regression. RESULTS: All eyes achieved significant reduction of IOP and number of medications with a final IOP of 15±3.2mm Hg at a mean follow-up of 8±32. months. While mild scarring was seen more common in MIT, severe scarring was seen in >65% of GATT eyes compared to 31% of MIT eye, p<0.001. An open saucer was equally seen in MIT and GATT while the trench pattern was more commonly seen in GATT eyes (>50%). Severe scarring in a trench pattern seemed to predict the need for medications for IOP control, though they independently did not seem to influence the final IOP or surgical outcome. CONCLUSION: A severe form of scarring in a trench pattern on AS-OCT predicted the need for glaucoma medications after MIGS surgery. Regular monitoring of the scarring responses by AS-OCT and clinical examination are necessary to identify those at need for medications after MIGS.

'Intraoperative predictors for clinical outcomes after microinvasive glaucoma surgery".

PURPOSE: To evaluate the clinical applicability of intraoperative predictors for surgical outcomes after gonioscopy-assisted transluminal trabeculotomy (GATT) and microincisional trabeculectomy (MIT). METHODS: Consecutive patients with primary, or secondary glaucoma (trauma, aphakic, or status post-retinal surgeries) with uncontrolled IOP>21mm Hg, who were scheduled to undergo GATT or MIT with or without significant cataract surgery, at a tertiary eye centre in East India between September 2021 to March 2023, were included. All surgeries were done by a single surgeon. Blanching and Trypan blue (0.4%) staining after intracameral injection using a 25 canula, were analysed in each video. The extent/pattern of blanching and blue staining in each eye was analysed objectively using an overlay of a circle with 12 sectors and a protractor tool to quantify the degrees or quadrants of blanching/staining. Multivariate regression was used to identify predictors for surgical success or the need for medications after surgery. RESULT: Of 167 eyes that were included (male: female- 134: 33), 49 eyes and 118 eyes underwent GATT and MIT, respectively, with 81 of 167 eyes undergoing concurrent cataract surgery. All eyes had a significant reduction in the number of medications after surgery. Blanching was seen in 154 of 167 eyes in a mean of 2±1.8 quadrants with 41% of eyes showing a blanching effect in >3 quadrants. Of 99 of 167 eyes where Trypan blue staining was assessed, staining in a venular, diffuse haze, or reticular pattern of staining was seen in 73 eyes, 26 eyes showed blue staining in >2 quadrants, with 16% staining in >3 quadrants. Surgical success was not predicted by the quadrants of blanching, blue staining, or other clinical variables (age, visual field, baseline intraocular pressure, type of surgery). The variables significantly predicting the need for medications included blanch (r = -0.1, p = 0.03), and blue staining (r = -0.1, p = 0.04) in <2 quadrants. CONCLUSIONS: Blanching and Trypan blue staining in >2 quadrants after GATT or MIT can serve as surrogate predictors for the need for medications. However more studies are mandated to find predictors for surgical success after GATT or MIT.

Tarlatamab for Patients with Previously Treated Small-Cell Lung Cancer.

BACKGROUND: Tarlatamab, a bispecific T-cell engager immunotherapy targeting delta-like ligand 3 and CD3, showed promising antitumor activity in a phase 1 trial in patients with previously treated small-cell lung cancer. METHODS: In this phase 2 trial, we evaluated the antitumor activity and safety of tarlatamab, administered intravenously every 2 weeks at a dose of 10 mg or 100 mg, in patients with previously treated small-cell lung cancer. The primary end point was objective response (complete or partial response), as assessed by blinded independent central review according to the Response Evaluation Criteria in Solid Tumors, version 1.1. RESULTS: Overall, 220 patients received tarlatamab; patients had previously received a median of two lines of treatment. Among patients evaluated for antitumor activity and survival, the median follow-up was 10.6 months in the 10-mg group and 10.3 months in the 100-mg group. An objective response occurred in 40% (97.5% confidence interval [CI], 29 to 52) of the patients in the 10-mg group and in 32% (97.5% CI, 21 to 44) of those in the 100-mg group. Among patients with an objective response, the duration of response was at least 6 months in 59% (40 of 68 patients). Objective responses at the time of data cutoff were ongoing in 22 of 40 patients (55%) in the 10-mg group and in 16 of 28 patients (57%) in the 100-mg group. The median progression-free survival was 4.9 months (95% CI, 2.9 to 6.7) in the 10-mg group and 3.9 months (95% CI, 2.6 to 4.4) in the 100-mg group; the estimates of overall survival at 9 months were 68% and 66% of patients, respectively. The most common adverse events were cytokine-release syndrome (in 51% of the patients in the 10-mg group and in 61% of those in the 100-mg group), decreased appetite (in 29% and 44%, respectively), and pyrexia (in 35% and 33%). Cytokine-release syndrome occurred primarily during treatment cycle 1, and events in most of the patients were grade 1 or 2 in severity. Grade 3 cytokine-release syndrome occurred less frequently in the 10-mg group (in 1% of the patients) than in the 100-mg group (in 6%). A low percentage of patients (3%) discontinued tarlatamab because of treatment-related adverse events. CONCLUSIONS: Tarlatamab, administered as a 10-mg dose every 2 weeks, showed antitumor activity with durable objective responses and promising survival outcomes in patients with previously treated small-cell lung cancer. No new safety signals were identified. (Funded by Amgen; DeLLphi-301 ClinicalTrials.gov number, NCT05060016.).

'Microincisional trabeculectomy for glaucoma".

PURPOSE: To evaluate the short-term clinical outcomes of microincisional trabeculectomy (MIT), a new technique of ab-interno trabeculectomy. METHODS: Consecutive patients with open-angle glaucoma identified from the hospital database that underwent MIT with or without cataract surgery between September 2021 to June 2022 at a tertiary eye centre in East India, were screened. Those with a follow-up of < 6 months or with incomplete data were excluded. MIT was done ab-interno using microscissors and microforceps in 2-4 clock hours of the nasal angle via a temporal incision. The intraocular pressure (IOP) reduction at 6 months, and reduction in the number of medications after surgery were analysed. Surgical success (IOP>6 and <22 mm Hg), complications, angle features on anterior segment optical coherence tomography (ASOCT), and the need for additional surgeries were analysed. RESULTS: We included thirty-two eyes of 32 patients with open-angle glaucoma (including n = 9 eyes that underwent concurrent cataract surgery) with a preoperative mean IOP of 22 ±11.1 mm Hg and visual field index of 47±37.9%. All eyes achieved >30% IOP reduction, with a final IOP of 14±6.9 mm Hg at 6 months. Surgical success in 31 of 32 eyes with complete success seen in 28 eyes with none of the eyes requiring >1 medication for IOP control. Hyphema was seen in 4 eyes, while transient IOP spikes at 1 day-1 month were seen in 5 eyes, none of which required any additional interventions. One eye with persistent raised IOP at 1 month required incisional trabeculectomy for uncontrolled IOP with 2 medications. CONCLUSION: MIT, a new technique of ab-interno trabeculectomy, is effective in terms of IOP control and reduction in the number of medications while having fewer complications. Long-term studies comparing the efficacy of MIT with incisional trabeculectomy, or other procedures are warranted in the future.

Causes of Immediate and Early IOP Spikes After Circumferential Gonioscopy-Assisted Transluminal Trabeculotomy Using ASOCT.

Purpose: To report the early postoperative causes of intraocular pressure (IOP) spikes after complete circumferential gonioscopy-assisted transluminal trabeculotomy (GATT) using anterior segment optical coherence tomography (ASOCT). Methods: This was retrospective interventional case series of patients who underwent circumferential GATT by a single surgeon (APR) from 2021 to 2022 at a tertiary eye care in East India and who developed IOP spikes at 1 day-2 weeks (immediate) or early (>2 weeks-2 months) after GATT. The intraocular pressure (IOP), glaucoma medications, and angle evaluation by ASOCT at 1 week, and 1 month were compared between the two groups. Results: Thirty-two eyes of 32 consecutive patients, aged 40±20.1 years (20 exfoliation glaucoma, 9 juvenile open-angle, and 3 developmental glaucoma), that developed IOP spikes between immediate (n=20) or early (n=12) IOP spikes after GATT, were included. Immediate postoperative spikes were seen in 20 eyes due to retained viscoelastic (n=8), hyphema (n=8) in uncontrolled hypertension, and peripheral anterior synechiae (PAS) (n=6) of which only one eye required medication at 2 months. Causes of immediate IOP spikes on ASOCT included an inverted/reattached trabecular shelf, iris strands in eyes with abnormal iris in developmental glaucoma, and PAS in 1-2 quadrants. Causes of early IOP spikes included PAS >3 quadrants (n=8), and hyperreflective fibrotic tissue over TM (n=4) with 50% requiring medications at 2 months. The ASOCT features predicting the need for medications were PAS >3 quadrants (OR=8.4[2.2-14.3], p=0.03) and fibrotic TM closure (OR=5.4, [3.6-10.2], p=0.003). One eye with macrohyphema 3 days after surgery owing to uncontrolled hypertension (owing to the stoppage of medicines), required additional incisional trabeculectomy within 1 month of surgery. Conclusion: Immediate IOP spikes are mostly self-resolving as opposed to early IOP spikes >2 weeks that require medications after GATT. Gonioscopic PAS >3 quadrants, and fibrotic TM closure were the main identifiable ASOCT causes predicting the need for medications after GATT.

Tarlatamab, a First-in-Class DLL3-Targeted Bispecific T-Cell Engager, in Recurrent Small-Cell Lung Cancer: An Open-Label, Phase I Study.

PURPOSE: Small-cell lung cancer (SCLC) is an aggressive malignancy with limited treatments. Delta-like ligand 3 (DLL3) is aberrantly expressed in most SCLC. Tarlatamab (AMG 757), a bispecific T-cell engager molecule, binds both DLL3 and CD3 leading to T-cellb-mediated tumor lysis. Herein, we report phase I results of tarlatamab in patients with SCLC. PATIENTS AND METHODS: This study evaluated tarlatamab in patients with relapsed/refractory SCLC. The primary end point was safety. Secondary end points included antitumor activity by modified RECIST 1.1, overall survival, and pharmacokinetics. RESULTS: By July 19, 2022, 107 patients received tarlatamab in dose exploration (0.003 to 100 mg; n = 73) and expansion (100 mg; n = 34) cohorts. Median prior lines of anticancer therapy were 2 (range, 1-6); 49.5% received antiprogrammed death-1/programmed death ligand-1 therapy. Any-grade treatment-related adverse events occurred in 97 patients (90.7%) and grade b % 3 in 33 patients (30.8%). One patient (1%) had grade 5 pneumonitis. Cytokine release syndrome was the most common treatment-related adverse event, occurring in 56 patients (52%) including grade 3 in one patient (1%). Maximum tolerated dose was not reached. Objective response rate was 23.4% (95% CI, 15.7 to 32.5) including two complete and 23 partial responses. The median duration of response was 12.3 months (95% CI, 6.6 to 14.9). The disease control rate was 51.4% (95% CI, 41.5 to 61.2). The median progression-free survival and overall survival were 3.7 months (95% CI, 2.1 to 5.4) and 13.2 months (95% CI, 10.5 to not reached), respectively. Exploratory analysis suggests that selecting for increased DLL3 expression can result in increased clinical benefit. CONCLUSION: In patients with heavily pretreated SCLC, tarlatamab demonstrated manageable safety with encouraging response durability. Further evaluation of this promising molecule is ongoing.

DNA-Bound p53-DNA-Binding Domain Interconverts between Multiple Conformations: Implications for Partner Protein Recognition.

Protein-protein interaction networks are critical components of cellular regulation. Hub proteins, defined by their ability to interact with numerous protein partners, are the pivots of these networks. A hypothesis that an ensemble of rapidly interconverting conformational states contributes significantly to the ability of hub proteins to interact with diverse partners has been proposed. The master gene regulator p53 is a prototype multidomain hub protein. Its DNA-binding domain alone is involved in interactions with many of its partner proteins. We investigated the dynamics of the p53 DNA-binding domain by 15N-NMR Carr-Purcell-Meiboom-Gill relaxation methods. In the DNA-bound state, we detected conformational exchanges in the domain in the microsecond to millisecond timescale, while dynamics at this timescale was not detectable in the free state. This suggests that the binding of p53 to specific DNA sequences promotes exchange between two or more conformational states, creating a broad conformational repertoire necessary for interacting with many partner proteins.

Structure, dynamics and lipid interactions of serotonin receptors: excitements and challenges.

Serotonin (5-hydroxytryptamine, 5-HT) is an intrinsically fluorescent neurotransmitter found in organisms spanning a wide evolutionary range. Serotonin exerts its diverse actions by binding to distinct cell membrane receptors which are classified into many groups. Serotonin receptors are involved in regulating a diverse array of physiological signaling pathways and belong to the family of either G protein-coupled receptors (GPCRs) or ligand-gated ion channels. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions such as sleep, mood, pain, anxiety, depression, aggression, and learning. Serotonin receptors act as drug targets for a number of diseases, particularly neuropsychiatric disorders. The signaling mechanism and efficiency of serotonin receptors depend on their amazing ability to rapidly access multiple conformational states. This conformational plasticity, necessary for the wide variety of functions displayed by serotonin receptors, is regulated by binding to various ligands. In this review, we provide a succinct overview of recent developments in generating and analyzing high-resolution structures of serotonin receptors obtained using crystallography and cryo-electron microscopy. Capturing structures of distinct conformational states is crucial for understanding the mechanism of action of these receptors, which could provide important insight for rational drug design targeting serotonin receptors. We further provide emerging information and insight from studies on interactions of membrane lipids (such as cholesterol) with serotonin receptors. We envision that a judicious combination of analysis of high-resolution structures and receptor-lipid interaction would allow a comprehensive understanding of GPCR structure, function and dynamics, thereby leading to efficient drug discovery.

Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock.

The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.

Comprehensive structural modeling and preparation of human 5-HT2A G-protein coupled receptor in functionally active form.

The serotonin 2A receptor (5-HT2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT2A R has marked distinction with ligand-bound conformations of 5-HT2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier.

Parity of access to memory services in London for the BAME population: a cross-sectional study.

OBJECTIVE: To investigate whether referrals to memory services in London reflect the ethnic diversity of the population. METHODS: Memory service data including referral rates of BAME was collected from London Clinical Commissioning Groups (CCGs). RESULTS: The expected percentage of BAME referrals using census data was compared against White British population percentages using the chi squared test. We found that within 13,166 referrals to memory services across London, the percentage of people from BAME groups was higher than would be expected (20.3 versus 19.4%; χ2 = 39.203, d.f. = 1, p < 0.0001) indicating that generally people from BAME groups are accessing memory services. Seventy-nine percent of memory services had more referrals than expected or no significant difference for all BAME groups. When there were fewer referrals then expected, the largest difference in percentage for an individual ethnic group was 3.3%. CONCLUSIONS: Results are encouraging and may indicate a significant improvement in awareness of dementia and help seeking behaviour among BAME populations. Prevalence of dementia in some ethnic groups may be higher so these numbers could still indicate under-referral. Due to the data available we were unable to compare disease severity or diagnosis type.

Conformational Entropy as a Determinant of the Thermodynamic Stability of the p53 Core Domain.

Mutations in the core domain of tumor suppressor protein p53 have been associated with ∼50% of the occurrences of human cancers. A majority of these mutations inactivate p53 function by destabilizing its native structure. Although studies have shown p53's function can be restored by stabilizing the mutants to their wild-type conformation with immense therapeutic potential, its applicability has been restricted because of our limited understanding of the precise nature of destabilization arising from changes in the mutant p53's structure and dynamics. Here, using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations, we have probed the conformational flexibility in three of the most widespread and clinically important "hot spot" mutants of the p53 core domain. Our results show that NMR order parameter-derived conformational entropy is linearly correlated with the change in free energy of urea-mediated denaturation, the latter being a well-established reporter of stability in p53 core domain mutants. Using a linear regression function, we show that the three parameters of equilibrium denaturation experiments, i.e., the free energy of denaturation (Δ GD-NH2O), the slope of the transition ( m), and the urea concentration at 50% denaturation ([urea]50%), can be used to predict the conformational entropy in p53 core domain mutants, thereby demonstrating a method for using these parameters as predictors of a protein's conformational entropy, which has been known to shape the functional properties of proteins.

Skeletal Muscle Dystrophy mutant of lamin A alters the structure and dynamics of the Ig fold domain.

Mutations in the different domains of A-type lamin proteins cause a diverse plethora of diseases collectively termed as laminopathies which can affect multiple organs. Ig fold is one such domain of lamin A which is implicated in numerous nuclear interactions wherein the mutations lead to different laminopathies. W514R is one such mutation in the Ig fold which leads to severe phenotypes in Skeletal Muscle Dystrophy (SMD) which is a class of laminopathies. In this report, we elucidated gross alterations in structure and dynamics at the level of individual amino acids. These studies indicate altered conformational features of residues in the close vicinity of W514. Imaging of mammalian cells transfected with the mutant have shown distinct perturbation of the nuclear meshwork with concomitant alteration in nuclear interactions as a result of increased oligomerization of Ig W514R. Hence, this novel approach of amalgamating theoretical and experimental procedures to predict the severity of a mutant in the context of laminopathies could be extended for numerous lamin A mutants.

Cloning, Expression, Purification and Characterization of Oligomeric States of the Native 5HT2A G-Protein-Coupled Receptor.

BACKGROUND: The 5HT2A G-Protein Coupled Receptor (GPCR) is an important family of receptors involved in an array of neuromodulatory functions. Their dysregulation has been implicated in a number of psychiatric diseases. In spite of the importance of this GPCR, high resolution structure and mechanistic details of its function is unknown. Cholesterol plays an important role in the function of many receptors and reduced cholesterol levels can lead to disruption of serotonergic pathways. However, the role of cholesterol in the formation of GPCR oligomers has not been previously shown for this receptor. Given that receptor dimers have been shown to be the functional unit of this receptor, it is important to investigate the effect of cholesterol in the oligomeric state of 5HT2A receptor. OBJECTIVES: The main objective of this work is to clone, over-express and purify the 5HT2A receptor and investigate the effect of cholesterol in its oligomer formation. METHODS: The 5HT2A receptor (5HT2AR) DNA construct was subcloned into pFastBac-HT vector and the purified bacmid was used to transfect healthy Sf9 cells. After subsequent passages, a high titer baculovirus was used for over-expression in Sf9 cells. To verify whether the over-expressed receptor was localized in the membrane or cytosolic fraction, cells with and without baculoviral infection were analyzed by immunocytochemistry. Subsequently, the over-expression conditions required to obtain sufficient quantity of the receptor was optimized followed by the optimization of the purification conditions. Finally, the culture was scaled up and the receptor was purified by affinity chromatography. The over-expression of the receptor was checked by Western blotting and purity was analyzed by Coomassie stained SDS PAGE. Cryo-electron microscopy experiments were performed on the purified receptor in presence and absence of cholesterol and at multiple concentrations to rule out any concentration dependent effect on the oligomer formation. RESULTS: Immunocytochemistry experiments showed prominent nuclear staining; however, bright green staining along the cell membrane was observed only for the infected cells, suggesting appropriate trafficking of majority of the over-expressed receptors to the cell membrane. Results of cryoelectron microscopy show that the receptor with cholesterol had particles that were bigger in size (~11 - 12 nm) compared to the dimension of known GPCR homologs. In contrast, the receptor after removal of cholesterol revealed a uniform distribution of smaller particles (~5 - 6 nm) that is approximately half the size of 5HT2AR particles with cholesterol. Comparing the 2D average views of detergent-encapsulated 5HT2AR particles with the overall dimensions of other 5HT receptor analogs, we show that while a 5HT2AR dimer more closely matches the dimensions of particles with CHS, only a monomer can be fit to particles without CHS. Importantly, even at higher receptor concentration and particle density, the size for 5HT2AR particles without CHS remains the same, suggesting that dimerization is unlikely an effect of concentration. CONCLUSION: Our results indicated that 5HT2A receptor primarily forms a dimer in presence of cholesterol whereas it predominantly forms a monomer when cholesterol is removed.

Comparing ganglion cell-inner plexiform layer thickness with focal and global responses on multifocal electroretinogram in glaucoma.

BACKGROUND: The aim of this study was to evaluate responses on multifocal electroretinogram (mfERG) with ganglion cell-inner plexiform layer (GCIPL) thickness on cirrus spectral-domain optical coherence tomography (SD-OCT) in glaucoma. METHODS: All diagnosed glaucoma patients attending glaucoma services at our institute from November 2012 to April 2013 were screened for this observational hospital-based study. Controls included patients attending our outpatient services for general eye checkup. Structural parameters on SD-OCT including GCIPL and retinal nerve fiber layer (RNFL) thickness were compared with functional parameters on mfERG in early (mean deviation <-6 dB), moderate (-6 to - 12 dB), and controls. RESULTS: A total of 54 cases and 33 controls fulfilling inclusion criteria were recruited for the study. The average and minimum GCIPL thickness did not vary significantly between early and control eyes while moderate glaucoma eyes had marginally lower GCIPL thickness than early glaucoma eyes. The GCIPL minimum thickness on univariate regression was found to be influenced by N2 amplitudes (ß = -0.5, P = 0.012) and global N2P1amplitudes (ß =0.6, P = 0.01) in moderate glaucoma. In early glaucoma, these were influenced only by RNFL parameters with no association with functional mfERG responses. Multivariate logistic regression identified global N2P1 amplitude to be significantly influencing GCIPL average and minimum thickness (P = 0.01 and 0.02, R2 = 47.8% and 52.3%, respectively) in moderate glaucoma. Maximum area under the curve was found for GCIPL minimum (95% confidence interval [CI] 0.53-0.81) and N2P1 amplitude (95% CI 0.55-0.80). CONCLUSIONS: The second order responses N2P1 and N2 amplitude on mfERG predict function that correlated with structural GCIPL thickness in moderate glaucoma. Early glaucoma may be best predicted by RNFL thickness rather than on mfERG responses.

Effect of amino acid mutations on the conformational dynamics of amyloidogenic immunoglobulin light-chains: A combined NMR and in silico study.

The conformational dynamics of a pathogenic κ4 human immunoglobulin light-chain variable domain, SMA, associated with AL amyloidosis, were investigated by 15N relaxation dispersion NMR spectroscopy. Compared to a homologous light-chain, LEN, which differs from SMA at eight positions but is non-amyloidogenic in vivo, we find that multiple residues in SMA clustered around the N-terminus and CDR loops experience considerable conformational exchange broadening caused by millisecond timescale protein motions, consistent with a destabilized dimer interface. To evaluate the contribution of each amino acid substitution to shaping the dynamic conformational landscape of SMA, NMR studies were performed for each SMA-like point mutant of LEN followed by in silico analysis for a subset of these proteins. These studies show that a combination of only three mutations located within or directly adjacent to CDR3 loop at the dimer interface, which remarkably include both destabilizing (Q89H and Y96Q) and stabilizing (T94H) mutations, largely accounts for the differences in conformational flexibility between LEN and SMA. Collectively, our studies indicate that a correct combination of stabilizing and destabilizing mutations is key for immunoglobulin light-chains populating unfolded intermediates that result in amyloid formation, and underscore the complex nature of correlations between light-chain conformational flexibility, thermodynamic stability and amyloidogenicity.

Intrinsic Differences in Backbone Dynamics between Wild Type and DNA-Contact Mutants of the p53 DNA Binding Domain Revealed by Nuclear Magnetic Resonance Spectroscopy.

Mutations in p53's DNA binding domain (p53DBD) are associated with 50% of all cancers, making it an essential system to investigate and understand the genesis and progression of cancer. In this work, we studied the changes in the structure and dynamics of wild type p53DBD in comparison with two of its "hot-spot" DNA-contact mutants, R248Q and R273H, by analysis of backbone amide chemical shift perturbations and 15N spin relaxation measurements. The results of amide chemical shift changes indicated significantly more perturbations in the R273H mutant than in wild type and R248Q p53DBD. Analysis of 15N spin relaxation rates and the resulting nuclear magnetic resonance order parameters suggests that for most parts, the R248Q mutant exhibits limited conformational flexibility and is similar to the wild type protein. In contrast, R273H showed significant backbone dynamics extending up to its ß-sandwich scaffold in addition to motions along the DNA binding interface. Furthermore, comparison of rotational correlation times between the mutants suggests that the R273H mutant, with a higher correlation time, forms an enlarged structural fold in comparison to the R248Q mutant and wild type p53DBD. Finally, we identify three regions in these proteins that show conformational flexibility to varying degrees, which suggests that the R273H mutant, in addition to being a DNA-contact mutation, exhibits properties of a conformational mutant.

Occurrence of COPD in Patients with Respiratory Allergy: A Clinico-Spirometric Evaluation in a Tertiary Hospital, Kolkata.

INTRODUCTION: Smoking is established as the most important causative factor responsible for Chronic Obstructive Pulmonary Disease (COPD). Occurrence of allergy in COPD patients causes acute exacerbation of this disease, but role of allergy is not established in aetiopathogenesis of COPD. AIM: The present study was aimed at evaluation of occurrence of COPD in patients having symptoms suggestive of respiratory allergy. MATERIALS AND METHODS: An observational cross-sectional study was conducted to evaluate occurrence of COPD in patients having respiratory allergic symptoms by routine spirometric screening. Five hundred and fifty urban patients aged 18-60 years (both gender) ailing from chronic respiratory symptoms like cough, wheeze and Shortness Of Breath (SOB), who were referred from OPDs of RGKMCH, Kolkata, were included in this study. After obtaining detailed clinical profile, patients were divided into two groups: subjects having additional clinical symptoms suggestive of respiratory allergy (n=260) like nasal catarrh, nasal stuffiness and sneezing and subjects with no symptoms suggestive of respiratory allergy (n=290). Thereafter, routine spirometry was carried out following recommendations of ATS/ERS (2005). Patients were then categorized based on FVC, FEV1, FEV1/FVC, FEF25-75 and PEFR percent predicted values. RESULTS: Study revealed that 18.97% of non-allergic population was suffering from COPD whereas only 7.69% of allergic subjects had COPD. This difference was statistically highly significant (p=0.0001). Although there was no significant difference in prevalence of respiratory symptoms between these two groups. CONCLUSION: Present study concludes that patients with respiratory allergy may have coexistent COPD but occurrence of COPD is much less than that in patients with no respiratory allergy.

Correlation of macular structure and function in a boy with primary foveomacular retinitis and sequence of changes over 5 years.

PURPOSE: To describe the clinical characteristics, macular structure and function, and to document sequential changes over 5 years in a 10-year-old boy with bilateral primary foveomacular retinitis. METHODS: A 10-year-old boy presented with sudden onset scotoma in both eyes, experienced after getting up from bed on a non-eclipse day. He persistently denied direct sun-gazing. He neither had any significant systemic illness, nor was using any medications. In addition to a detailed examination at presentation that included fundus fluorescein angiogram (FFA), electroretinogram (ERG), pattern ERG and electrooculogram (EOG), he was examined periodically for 5 years with Humphrey visual field (HVF), spectral domain optical coherence tomogram (SDOCT), Amsler grid charting and multifocal ERG. The macular structure and functions were analyzed over the years and correlated with the symptoms. RESULTS: All findings were bilaterally symmetrical at each visit. At presentation, his corrected visual acuity was 20/25 with subfoveal yellow dot similar to solar retinopathy, central scotoma with reduced foveal threshold in HVF 24-2, micropsia in Amsler grid, missing of two plates on Ishihara color vision chart, transfoveal full thickness hyper-reflective band on SD OCT, unremarkable FFA and normal foveal peak in mfERG. The flash ERG and EOG were unremarkable. A month later, his VA improved to 20/20, he had relative scotoma in Amsler grid, no scotoma in HVF (10-2), restoration of the inner segment of the photoreceptors with sharp defect involving ellipsoid and photoreceptor interdigitation zone in SDOCT and blunting of foveal peaks in mfERG. Three months later, his corrected VA was 20/20 with relative scotoma in Amsler grid, normal color vision, no scotoma in HVF 10-2 and unchanged SDOCT findings. In subsequent examinations at 6, 9, 14, 29, 39 and 60 months, he was symptomless with VA 20/20, unremarkable fundus, normal Amsler grid and HVF (normal foveal threshold), unchanged SDOCT findings and the reduced foveal peaks on mfERG in both eyes got normalized only at 60 months. CONCLUSION: Presented here is a case of bilaterally symmetrical idiopathic foveomacular retinitis that had a clinical appearance similar to solar retinopathy. The fundus changes persisted for 4 weeks, the symptoms and changes in Amsler grid lasted for 3 months, and the foveal threshold in visual fields normalized within 3 months. Maximum change in the SDOCT defect occurred within a month, and the extrafoveal defect in the ellipsoid and photoreceptor interdigitation line persisted despite resolution of symptoms and resolution of the visual field defect and normal distance vision. Probably, the foveal lesion detected on SDOCT was too small to cause a reduction in the distance visual acuity or show up in the visual field and mfERG later.

Vessel Caliber in Normal Tension and Primary Open Angle Glaucoma Eyes With Hemifield Damage.

PURPOSE OF THE STUDY: The purpose of the study was to evaluate retinal vessel diameter in age-matched normal tension glaucoma (NTG) and primary open angle glaucoma (POAG) eyes with hemifield involvement. METHODS: Fundus photographs of patients with hemifield defect, good visibility of retinal nerve fiber layer defect, and vessels were compared with 30 controls. One eye of each patient (28 NTG and 30 POAG) was randomly chosen for analysis using Image J software by different clinicians at 2 levels. The structural parameters analyzed included retinal nerve fiber layer defect width, diameter of vessels (superotemporal or inferotemporal artery and superotemporal or inferotemporal vein). RESULTS: The average superotemporal artery diameter was similar in NTG (71±16.8 µm), POAG (79±26.6 µm), and controls (82±14.1 µm), P=0.2 with similar pattern seen for other vessels. The affected quadrant in all eyes and those with disc hemorrhage (n=8) did not have significantly different arteries and veins diameter as compared with the unaffected quadrant in that eye. No relation of artery or vein diameter with retinal nerve fiber layer defect width or clinical variables in NTG or POAG eyes was seen on multivariate regression analysis. CONCLUSIONS: The vessel diameter was not significantly different in the affected and unaffected quadrants of age and severity-matched NTG and POAG eyes with hemifield structural and functional defect as reported in earlier studies. These results point toward the possibility that vascular diameter changes may not be the cause for glaucomatous changes.