Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

A Tag-Free Platform for Synthesis and Screening of Cyclic Peptide Libraries.

In the realm of high-throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post-screening. Our innovation introduces a tag-free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity-enhancing derivatization method, utilized in tandem with nano LC-MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

Accurate de novo design of hyperstable constrained peptides.

Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18-47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N-C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.

Clinical presentations and surgical outcomes of intraocular foreign body presenting to an ocular trauma unit.

OBJECTIVES: To describe, evaluate, and identify the characteristics, prognostic factors, and visual outcomes in patients with intraocular foreign body (IOFB) in a Latin American population. METHODS: A retrospective, observational case-series of patients with a diagnosis of IOFB. Variables analyzed included age, gender, initial and final best correct visual acuity (BCVA), ocular trauma score, intraocular pressure, mechanism of injury, material and number of IOFB, zone of injury, timing of primary repair and IOFB removal, complications, and follow up. RESULTS: Sixty-one patients with IOFB were identified of which 97% were male with a mean age of 37.9 years (SD 2.16). The most common IOFB location was intravitreal (43%). IOFBs were metallic in 78%, vegetal in 3%, and other materials in 11%. Primary repair and secondary IOFB removal were performed at a mean timepoint of 3 days and 5 days, respectively. Systemic and topical antibiotics were administered to all patients. The initial BCVA was 1.62 logMAR and the final was 0.6 logMAR, which was statistically significant (Pearson's chi-squared test, p value 0.01). No cases of endophthalmitis were seen. CONCLUSION: IOFB removal can be delayed when there are no signs of infection or evidence of retinal detachment, without an increased risk of endophthalmitis and a negative impact on visual outcomes. Use of topical and systemic antibiotics appear sufficient to prevent endophthalmitis in these cases.

Diabetic myonecrosis in end-stage renal disease.

Diabetic myonecrosis or muscle infarction (DMI), a clinicoradiological entity is an underdiagnosed complication of diabetes mellitus. It refers to spontaneous aseptic necrosis of skeletal muscles commonly of the lower limb without evidence of any large vessel disease. It presents as painful swollen limb without any external insult in patients with long-standing diabetes mellitus with other microvascular complications especially nephropathy. We present four instances of DMI in our patients who had end-stage renal disease with a varied course.

MANAGEMENT OF GIANT RETINAL TEARS USING TRANSSCLERAL DIODE LASER RETINOPEXY AND SHORT-TERM POSTOPERATIVE TAMPONADE WITH PERFLUORO-N-OCTANE.

PURPOSE: To determine the results of pars plana vitrectomy for giant retinal tear detachments using transscleral diode laser retinopexy and short-term postoperative tamponade with perfluoro-n-octane (PFnO). METHODS: Twenty consecutive patients with fresh giant retinal tears were enrolled in a single-arm prospective study. One case was withdrawn for technical reasons. The remainder all underwent pars plana vitrectomy, PFnO injection, transscleral diode laser retinopexy to the edge of the giant retinal tear, and short-term postoperative heavy liquid tamponade. None of the cases had scleral buckling or lensectomy. RESULTS: Nineteen cases (18 male and 1 female) with a mean age of 41 years (range 10-69 years) were followed up for a period of 6 months. Postoperative tamponade with PFnO was maintained for a mean of 7.6 days (range 4-21 days), after which it was exchanged for sulfur hexafluoride (SF6), perfluoropropane (C3F8) gas, or balanced salt solution. Final reattachment rate was 100%, with 3 (15.7%) patients requiring additional surgery. Best-corrected visual acuity at final follow-up was 20/40 or better in 11 eyes (58%), between 20/60 and 20/200 in 7 (37%), and 20/400 in 1 (5%). CONCLUSION: In this series of acute giant retinal tears, transscleral diode laser retinopexy together with the use of PFnO for short-term postoperative tamponade achieved excellent anatomical and visual results.

Acute kidney injury is associated with higher mortality and healthcare costs in hospitalized patients with cirrhosis.

INTRODUCTION AND OBJECTIVES: AKI is known to be associated with increased risk of mortality, however limited information is available on how AKI impacts healthcare costs and resource utilization in hospitalized patients with cirrhosis. Previous studies have had variable definitions of AKI, resulting in inconsistent reporting of the true impact of AKI in patients with cirrhosis. METHODS: Data from the Nationwide Inpatient Sample (NIS) which contains data from 44 states and 4378 hospitals, accounting for over 7 million discharges were analyzed. The inclusion data were all discharges in the 2012 NIS dataset with a discharge diagnosis of cirrhosis. RESULTS: A total of 32,605 patients were included in the analysis, incidence of AKI was 12.12% in patients with cirrhosis. Crude mortality was much higher for patients with cirrhosis and AKI (14.9% vs. 1.8%, OR 9.42, p<0.001) than for patients without AKI. In addition, mean LOS was longer (8.5 vs. 4.3 days, p<0.001) and median total hospital charges were higher for patients with AKI ($43,939 vs. $22,270, p<0.001). In multivariate logistic regression, controlling for covariates and mortality risk score, sepsis, ascites and SBP were predictors of AKI. CONCLUSIONS: AKI is relatively common in hospitalized patients with cirrhosis. Presence of AKI results in significantly higher inpatient mortality as well as LOS and resource utilization. Median hospitalization cost was twice as high in AKI patients. Early identification of patients at high risk for AKI should be implemented to reduce mortality and contain costs. Prognosis could be enhanced by utilizing biomarkers which could rapidly detect AKI.

Keeping an eye on syphilis

BACKGROUND: The objective of this article was to alert general practitioners (GPs) to the increase in ocular syphilis in the context of a worsening epidemic of syphilis among men who have sex with men (MSM). METHODS: This study used a retrospective case review of ocular syphilis cases that presented to the Royal Victorian Eye and Ear Hospital from January 2015 to August 2016. RESULTS: Twelve patients (19 eyes) were identified, including 11 males. The mean age was 35 years, and seven men were identified as MSM. Two men were diagnosed with human immunodeficiency virus (HIV) infection at presentation. Blurred vision (n = 10) and/or floaters (n = 9) were the most common presenting symptoms. All patients had uveitis as the manifestation of the ocular involvement; however, redness and pain were not universally reported. DISCUSSION: GPs should be alert to the possibility of ocular syphilis at the time of syphilis diagnosis, particularly among MSM. Urgent ophthalmic referral is required if the patient is found to have new onset visual symptoms.

Covalent Chemical Ligation Strategy for Mono- and Polyclonal Immunoglobulins at Their Nucleotide Binding Sites.

Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. Site-specific ligation of compounds (toxins or ligands) to antibodies has become increasingly important in the fields of therapeutic antibody-drug conjugates and bispecific antibodies. In this present study, we took advantage of the reported nucleotide-binding pocket (NBP) in the Fab arms of immunoglobulins by developing indole-based, 5-fluoro-2,4-dinitrobenzene-derivatized OBOC peptide libraries for the identification of affinity elements that can be used as site-specific derivatization agents against both mono- and polyclonal antibodies. Ligation can occur at any one of the few lysine residues located at the NBP. Immunoconjugates resulting from such affinity elements can be used as therapeutics against cancer or infectious agents.

Obesity paradox in advanced liver disease: obesity is associated with lower mortality in hospitalized patients with cirrhosis.

BACKGROUND & AIMS: To investigate how obesity impacts inpatient mortality, length of stay (LOS) and costs in patients with cirrhosis. Obesity is a growing epidemic associated with multiple co-morbidities, increased morbidity, and a significant economic burden on healthcare. Despite the overall harmful impact of obesity, the 'obesity paradox' has been described as decreased mortality among obese vs non-obese patients in various chronic medical conditions. METHODS: Analysis of the Nationwide Inpatient Sample (NIS) for 2012, which contains data from 44 states and 4378 hospitals. Data from all cases with primary, secondary or tertiary discharge diagnosis of cirrhosis identified by International Classification of Diseases-9 code 571.2, 571.5 571.6 were included. Primary outcomes included inpatient mortality, LOS, and hospital charges. Obesity as a predictor of mortality was defined by a predetermined obesity co-morbidity variable. RESULTS: A total of 32,605 patients were included. Crude mortality was lower for obese cirrhotic patients (2.7% vs 3.5%, P = 0.02) than for non-obese cirrhotic patients. In contrast, median LOS was longer (4 vs 3 days, P < 0.001) and median hospital charges were higher for obese cirrhotic patients ($26 803 vs $23 447, P < 0.001) In multivariate logistic regression, obesity was associated with a lower risk of inpatient mortality (OR=0.73, 95%CI: 0.55-0.95, P = 0.02). CONCLUSIONS: In the acute care setting, obese patients with cirrhosis have lower mortality than non-obese patients with cirrhosis, longer hospitalizations and higher healthcare cost, providing new evidence for the obesity paradox in cirrhosis. Obese cirrhotic patients are more likely to have enhanced nutritional reserve which may play a role in survival during acute illness.

Differentially expressed urinary biomarkers in children with idiopathic nephrotic syndrome.

BACKGROUND: We performed a discovery phase of urinary proteomic profile in children with idiopathic nephrotic syndrome and validated selected biomarkers. METHODS: Urinary proteomic profile was performed using isobaric tags for relative and absolute quantitation labeling, coupled with liquid chromatography-matrix assisted laser desorption and ionization analysis. Validation of biomarkers apolipoprotein A1, alpha 2 macroglobulin, orosomucoid 2, retinol binding protein 4 and leucine-rich alpha 2-glycoprotein 1 was done by enzyme-linked immunosorbent assay. RESULTS: Apolipoprotein A1 levels of <0.48 µg/mg of creatinine-differentiated steroid-resistant nephrotic syndrome (SRNS) from first episode nephrotic syndrome, area under curve (AUC) [0.99 (CI 0.9-1.0), 100 % sensitivity and 100 % specificity] and a value of <0.24 µg/mg of creatinine could differentiate SRNS from frequently relapsing nephrotic syndrome/steroid dependent nephrotic syndrome [AUC 0.99 (CI 0.9-1.0), 100 % sensitivity and 100 % specificity]. Alpha 2 macroglobulin could differentiate children with SRNS-focal segmental glomerulosclerosis (FSGS) from SRNS-minimal change disease (MCD) at values >3.3 µg/mg of creatinine [AUC 0.84 (CI 0.62-1.0), 90 % sensitivity and 85 % specificity]. Orosomucoid 2 >1.81 µg/mg of creatinine could distinguish SRNS-FSGS from SRNS-MCD [AUC 0.84 (CI 0.62-1.0), sensitivity 90 % and specificity 85.5 %]. RBP 4 value of >1.54 µg/mg of creatinine differentiated SRNS-FSGS from SRNS-MCD [AUC 0.87 (CI 0.68-1.0), sensitivity 90 % and specificity 85.7 %]. CONCLUSIONS: Lower level of apolipoprotein A1 in urine is suggestive of SRNS. Alpha 2 macroglobulin, retinol binding protein 4 and orosomucoid 2 are markers associated with FSGS, with alpha 2 macroglobulin being most predictive.

Expansive discovery of chemically diverse structured macrocyclic oligoamides.

Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

Reevaluation of the evolutionary events within recA/RAD51 phylogeny.

BACKGROUND: The recA/RAD51 gene family encodes a diverse set of recombinase proteins that affect homologous recombination, DNA-repair, and genome stability. The recA gene family is expressed across all three domains of life - Eubacteria, Archaea, and Eukaryotes - and even in some viruses. To date, efforts to resolve the deep evolutionary origins of this ancient protein family have been hindered by the high sequence divergence between paralogous groups (i.e. ~30% average pairwise identity). RESULTS: Through large taxon sampling and the use of a phylogenetic algorithm designed for inferring evolutionary events in highly divergent paralogs, we obtained a robust, parsimonious and more refined phylogenetic history of the recA/RAD51 superfamily. CONCLUSIONS: In summary, our model for the evolution of recA/RAD51 family provides a better understanding of the ancient origin of recA proteins and the multiple events that lead to the diversification of recA homologs in eukaryotes, including the discovery of additional RAD51 sub-families.

Assessment of adequate removal of ophthalmic viscoelastic device with irrigation/aspiration by quantifying intraocular lens 'Judders'.

BACKGROUND: To assess the efficacy of 'Judders' as a technique reflecting adequacy of removal of ophthalmic viscoelastic device in cataract surgery. DESIGN: Prospective, consecutive, single surgeon study. PARTICIPANTS: Cohort of 223 patients undergoing phacoemulsification. METHODS: 'Judders' are periodic, abrupt, horizontal displacements of the intraocular lens causing balanced salt solution to displace retropseudophakic ophthalmic viscoelastic device. The number of 'Judders' was correlated with axial length, anterior chamber depth, and preoperative and postoperative intraocular pressure. MAIN OUTCOME MEASURES: Number of 'Judders', axial length, anterior chamber depth, day 1 postoperative intraocular pressure. RESULTS: The mean number of 'Judders' was 3.2. There was a positive association between the number of 'Judders' and axial length, but not between number of 'Judders' and anterior chamber depth. Mean preoperative intraocular pressure was 14.5 mmHg; mean day 1 postoperative intraocular pressure was 15.6 mmHg. Intraocular pressure rose in 47% of cases. In six cases (5%), intraocular pressure rose greater than 10 mmHg (range 11-23 mmHg) from the preoperative level. CONCLUSIONS: Aspirating ophthalmic viscoelastic device with the irrigation/aspiration tip posterior to the intraocular lens may be associated with the risk of a posterior capsule tear. Maintaining the irrigation/aspiration tip anterior to the intraocular lens may offer a significant safety advantage. The number of 'Judders', usually 3-4, appears to be a safe and reliable end-point of complete ophthalmic viscoelastic device removal. There were significantly more 'Judders' in eyes with a longer axial length. The safety and efficacy of 'Judders' are reflected by the stable mean postoperative intraocular pressure.

Accommodative spasm and its different treatment approaches: A systematic review.

This article is about the accommodation spasm. The primary rule for near vision is ciliary muscle constriction, synchronised convergence of both eyes, and pupil constriction. Any weaknesses in these components could result in an accommodative spasm. Variable retinoscopic reflex, unstable refractive error, and lead of accommodation in near retinoscopy are common causes of spasm. We conducted a thorough literature search in the PubMed and Google Scholar databases for published journals prior to June 2022, with no data limitations. This review contains twenty-eight case reports, six cohort studies, four book references, four review articles, and two comparative studies after applying the inclusion and exclusion criteria. The majority of studies looked at accommodative spasm, near reflex spasm, and pseudomyopia. The most common causes of accommodative spasm are excessive close work, emotional distress, head injury, and strabismus. Despite side effects or an insufficient regimen, cycloplegic drops are effective in diagnosing accommodation spasm. The modified optical fogging technique is also effective and may be an option for treating accommodative spasm symptoms. Bifocals for near work, manifest refraction, base-in prisms, and vision therapy are some of the other management options. As a result, it requires a comprehensive clinical treatment strategy. This review aims to investigate the various aetiology and treatments responsible for accommodative spasm and proposes widely implementing the modified optical fogging method and vision therapy in clinics as comprehensive management to reduce the future upward trend of accommodative spasm.

The Effect of Illumination on Positive Fusional Vergence.

Background: Positive fusional vergence (PFV) is vital in maintaining fusion in critical and continuous near tasks such as reading or performing digital screen tasks. This study investigated how PFV changed under various lighting conditions. Methods: This cross-sectional study recruited 34 participants aged between 21 and 25 years, with best corrected visual acuity (BCVA) 0.0 logMAR and insignificant refractive error. Three different illuminations-low illumination (50 lux), medium lighting (100 lux), and high illumination (150 lux)-were used to examine the ocular parameters PFV (blur, break, and recovery points), contrast sensitivity and pupil diameter. Results: Pupil diameter changed significantly in different room illuminations (p = 0.00). There was no significant difference in contrast sensitivity across the three levels of room illumination (p = 0.368). Mean PFV (SD) (blur) was 14.5 (2.5) in 50 lux, 10.2 (2.2) in 100 lux, and 8.2 (2.1) in 150 lux. Under 50, 100 and 150 lux, respectively, the mean PFV (SD) (break) values were 16.7 (2.4), 13.4 (1.8), and 10.8 (2.2), and the mean PFV (SD) (recovery) values were 13.3 (2.1), 10.7 (2.1), and 7.5 (2.7). With increased illumination levels, PFV blur, break, and recovery values were significantly lower (p < 0.001). Conclusions: PFV values were significantly higher in lower illumination. Clinicians should be aware that room illumination affected the PFV values measured.

Cell-permeable chameleonic peptides: Exploiting conformational dynamics in de novo cyclic peptide design.

Membrane-traversing peptides offer opportunities for targeting intracellular proteins and oral delivery. Despite progress in understanding the mechanisms underlying membrane traversal in natural cell-permeable peptides, there are still several challenges to designing membrane-traversing peptides with diverse shapes and sizes. Conformational flexibility appears to be a key determinant of membrane permeability of large macrocycles. We review recent developments in the design and validation of chameleonic cyclic peptides, which can switch between alternative conformations to enable improved permeability through cell membranes, while still maintaining reasonable solubility and exposed polar functional groups for target protein binding. Finally, we discuss the principles, strategies, and practical considerations for rational design, discovery, and validation of permeable chameleonic peptides.

Cyclic peptide structure prediction and design using AlphaFold.

Deep learning networks offer considerable opportunities for accurate structure prediction and design of biomolecules. While cyclic peptides have gained significant traction as a therapeutic modality, developing deep learning methods for designing such peptides has been slow, mostly due to the small number of available structures for molecules in this size range. Here, we report approaches to modify the AlphaFold network for accurate structure prediction and design of cyclic peptides. Our results show this approach can accurately predict the structures of native cyclic peptides from a single sequence, with 36 out of 49 cases predicted with high confidence (pLDDT > 0.85) matching the native structure with root mean squared deviation (RMSD) less than 1.5 Å. Further extending our approach, we describe computational methods for designing sequences of peptide backbones generated by other backbone sampling methods and for de novo design of new macrocyclic peptides. We extensively sampled the structural diversity of cyclic peptides between 7-13 amino acids, and identified around 10,000 unique design candidates predicted to fold into the designed structures with high confidence. X-ray crystal structures for seven sequences with diverse sizes and structures designed by our approach match very closely with the design models (root mean squared deviation < 1.0 Å), highlighting the atomic level accuracy in our approach. The computational methods and scaffolds developed here provide the basis for custom-designing peptides for targeted therapeutic applications.

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor.

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.