Efficacy and Safety of Non-Vitamin-K Antagonist Oral Anticoagulants Versus Warfarin Across the Spectrum of Body Mass Index and Body Weight: An Individual Patient Data Meta-Analysis of 4 Randomized Clinical Trials of Patients With Atrial Fibrillation.

BACKGROUND: The efficacy and safety of non-vitamin-K antagonist oral anticoagulants (NOACs) across the spectrum of body mass index (BMI) and body weight (BW) remain uncertain. METHODS: We analyzed data from COMBINE AF (A Collaboration Between Multiple Institutions to Better Investigate Non-Vitamin K Antagonist Oral Anticoagulant Use in Atrial Fibrillation), which pooled patient-level data from the 4 pivotal randomized trials of NOAC versus warfarin in patients with atrial fibrillation. The primary efficacy and safety outcomes were stroke or systemic embolic events (stroke/SEE) and major bleeding, respectively; secondary outcomes were ischemic stroke/SEE, intracranial hemorrhage, death, and the net clinical outcome (stroke/SEE, major bleeding, or death). Each outcome was examined across BMI and BW. Because few patients had a BMI <18.5 kg/m2 (n=598), the primary analyses were restricted to those with a BMI ≥18.5 kg/m2. RESULTS: Among 58 464 patients, the median BMI was 28.3 (interquartile range, 25.2-32.2) kg/m2, and the median BW was 81.0 (interquartile range, 70.0-94.3) kg. The event probability of stroke/SEE was lower at a higher BMI irrespective of treatment, whereas the probability of major bleeding was lower at a higher BMI with warfarin but relatively unchanged across BMI with NOACs. NOACs reduced stroke/SEE overall (adjusted hazard ratio [HRadj], 0.80 [95% CI, 0.73-0.88]; P<0.001), with a generally consistent effect across BMI (Ptrend across HRs, 0.48). NOACs also reduced major bleeding overall (HRadj, 0.88 [95% CI, 0.82-0.94]; P<0.001), but with attenuation of the benefit at a higher BMI (trend test across BMI [Ptrend], 0.003). The overall treatment effects of NOACs versus warfarin for secondary outcomes were consistent across BMI, with the exception of the net clinical outcome and death. While these outcomes were overall reduced with NOACs (net clinical outcome, HRadj, 0.91 [95% CI, 0.87-0.95]; P<0.001; death, HRadj, 0.91 [95% CI, 0.86-0.97]; P=0.003), these benefits were attenuated at higher BMI (Ptrend, 0.001 and 0.08, respectively). All findings were qualitatively similar when analyzed across BW. CONCLUSIONS: The treatment effect of NOACs versus warfarin in atrial fibrillation is generally consistent for stroke/SEE across the spectrum of BMI and BW, whereas the reduction in major bleeding is attenuated in those with higher BMI or BW. Death and the net clinical outcome are overall reduced with NOACs over warfarin, although there remain uncertainties for these outcomes at a very high BMI and BW.

Sodium-Glucose Cotransporter-2 Inhibitors and Major Adverse Cardiovascular Outcomes: A SMART-C Collaborative Meta-Analysis.

BACKGROUND: Sodium-glucose cotransporter-2 inhibitors (SGLT2i) consistently improve heart failure and kidney-related outcomes; however, effects on major adverse cardiovascular events (MACE) across different patient populations are less clear. METHODS: This was a collaborative trial-level meta-analysis from the SGLT2i Meta-analysis Cardio-Renal Trialists Consortium, which includes all phase 3, placebo-controlled, outcomes trials of SGLT2i across 3 patient populations (patients with diabetes at high risk for atherosclerotic cardiovascular disease, heart failure [HF], or chronic kidney disease). The outcomes of interest were MACE (composite of cardiovascular death, myocardial infarction , or stroke), individual components of MACE (inclusive of fatal and nonfatal events), all-cause mortality, and death subtypes. Effect estimates for SGLT2i versus placebo were meta-analyzed across trials and examined across key subgroups (established atherosclerotic cardiovascular disease, previous myocardial infarction, diabetes, previous HF, albuminuria, chronic kidney disease stages, and risk groups). RESULTS: A total of 78 607 patients across 11 trials were included: 42 568 (54.2%), 20 725 (26.4%), and 15 314 (19.5%) were included from trials of patients with diabetes at high risk for atherosclerotic cardiovascular disease, HF, or chronic kidney disease, respectively. SGLT2i reduced the rate of MACE by 9% (hazard ration [HR], 0.91 [95% CI, 0.87-0.96], P<0.0001) with a consistent effect across all 3 patient populations (I2=0%) and across all key subgroups. This effect was primarily driven by a reduction in cardiovascular death (HR, 0.86 [95% CI, 0.81-0.92], P<0.0001), with no significant effect for myocardial infarction in the overall population (HR, 0.95 [95% CI, 0.87-1.04], P=0.29), and no effect on stroke (HR, 0.99 [95% CI, 0.91-1.07], P=0.77). The benefit for cardiovascular death was driven primarily by reductions in HF death and sudden cardiac death (HR, 0.68 [95% CI, 0.46-1.02] and HR, 0.86 [95% CI, 0.78-0.95], respectively) and was generally consistent across subgroups, with the possible exception of being more apparent in those with albuminuria (Pinteraction=0.02). CONCLUSIONS: SGLT2i reduce the risk of MACE across a broad range of patients irrespective of atherosclerotic cardiovascular disease, diabetes, kidney function, or other major clinical characteristics at baseline. This effect is driven primarily by a reduction of cardiovascular death, particularly HF death and sudden cardiac death, without a significant effect on myocardial infarction in the overall population, and no effect on stroke. These data may help inform selection for SGLT2i therapies across the spectrum of cardiovascular-kidney-metabolic disease.

Advances in synthesis and biological evaluation of CDK2 inhibitors for cancer therapy.

One of the leading causes of mortality in the world is cancer. This disease occurs when responsible genes that regulate the cell cycle become inactive due to internal or external factors. Specifically, the G1/S and S/G2 transitions in the cell cycle are controlled by a protein called cyclin-dependent kinase 2 (CDK2). CDKs, which play a crucial role in managing the cell cycle, have been a wide area of research in cancer treatment. Over the past 11 years, significant research has been made in identifying potent, targeted, and efficient inhibitors of CDK2. In this summary, we have summarized recent developments in the synthesis and biological evaluation of CDK2 inhibitors.

Evaluation and application of American College of Radiology Thyroid Imaging Reporting and Data System for improved malignancy detection in paediatric thyroid nodules.

OBJECTIVE: The American College of Radiology Thyroid Imaging Reporting and Data System (TI-RADS) is a widely used method for the management of adult thyroid nodules. However, its use in paediatric patients is controversial because adult fine needle aspiration biopsy (FNAB) recommendations may lead to delayed diagnoses of cancer in children. The objectives of this study were to evaluate the performance of TI-RADS in paediatric thyroid nodules and to tailor FNAB recommendations for children. METHODS: Consecutive surgically resected paediatric thyroid nodules from two tertiary care centres between 2003 and 2021 were reviewed. Ultrasounds were blindly scored by radiologists according to TI-RADS. Management recommendations based on TI-RADS were evaluated. Various modelling methodologies were used to determine the optimal cutoff for FNAB in children. RESULTS: Of the 96 patients, 79 (82%) were female and the median age at surgery was 16.1 years. Fifty (52%) nodules were malignant on surgical pathology. The area under the receiver operating characteristic curve of TI-RADS for predicting malignancy was 0.78. Adult TI-RADS recommendations would have resulted in 4% of cancerous nodules being lost to follow-up. Modifications to TI-RADS (FNAB of all TR3 nodules ≥1.5 cm, FNAB of TR4 and TR5 nodules ≥0.5 cm, surveillance of nodules ≥1 cm, consider surgery for nodules >4 cm) reduced this missed malignancy rate to 0%. CONCLUSIONS: TI-RADS can risk-stratify paediatric thyroid nodules. However, the system requires modifications to reduce the missed malignancy rate in paediatric thyroid nodules. Our data suggest that lower size thresholds for FNAB are warranted in children.

Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia.

This study presents the first messenger RNA (mRNA) therapy for metastatic ovarian cancer and cachexia-induced muscle wasting based on lipid nanoparticles that deliver follistatin (FST) mRNA predominantly to cancer clusters following intraperitoneal administration. The secreted FST protein, endogenously synthesized from delivered mRNA, efficiently reduces elevated activin A levels associated with aggressive ovarian cancer and associated cachexia. By altering the cancer cell phenotype, mRNA treatment prevents malignant ascites, delays cancer progression, induces the formation of solid tumors, and preserves muscle mass in cancer-bearing mice by inhibiting negative regulators of muscle mass. Finally, mRNA therapy provides synergistic effects in combination with cisplatin, increasing the survival of mice and counteracting muscle atrophy induced by chemotherapy and cancer-associated cachexia. The treated mice develop few nonadherent tumors that are easily resected from the peritoneum. Clinically, this nanomedicine-based mRNA therapy can facilitate complete cytoreduction, target resistance, improve resilience during aggressive chemotherapy, and improve survival in advanced ovarian cancer.

Evaluation of the atrial fibrillation better care pathway in the ENGAGE AF-TIMI 48 trial.

AIMS: The Atrial fibrillation Better Care (ABC) pathway is endorsed by guidelines to improve care of patients with atrial fibrillation (AF). However, whether the benefit of ABC pathway-concordant care is consistent across anticoagulants remains unclear. We assessed the association between ABC-concordant care and outcomes in this post hoc analysis from the ENGAGE AF-TIMI 48 trial, which was reported prior to the initial description of the ABC pathway. METHODS AND RESULTS: Patients were retrospectively classified as receiving ABC-concordant care based on optimal anticoagulation, adequate rate control, management of co-morbidities and lifestyle measures. Associations between ABC-concordance and outcomes were assessed with adjustment for components of the CHA2DS2-VASc and HAS-BLED scores. Of 20 926 patients, 7915 (37.8%) satisfied criteria of ABC-concordant care, which was associated with significantly lower incidence of stroke or systemic embolic event [stroke/SEE: hazard ratio (HRadj): 0.54; 95% confidence interval (CI): 0.47-0.63], major bleeding (HRadj 0.66; 95% CI: 0.58-0.75), major adverse cardiac events (HRadj 0.53; 95% CI: 0.48-0.58), primary net clinical outcome (composite of stroke/SEE, major bleeding or death; HRadj 0.61; 95% CI: 0.56-0.65), cardiovascular (CV) hospitalization (HRadj 0.78; 95% CI: 0.74-0.83), CV death (HRadj 0.52; 95% CI: 0.46-0.58), and all-cause mortality (HRadj 0.56; 95% CI: 0.51-0.62), P < 0.001 for each. These associations were qualitatively consistent for both edoxaban and warfarin and across patient subgroups. CONCLUSION: Atrial fibrillation Better Care pathway-concordant care is associated with reductions across multiple CV endpoints and all-cause mortality, with benefit in edoxaban- and warfarin-treated patients and across patient subgroups. Increasing implementation of ABC-concordant care may improve clinical outcomes of patients with AF irrespective of anticoagulant.

Matrix stiffness regulates lipid nanoparticle-mRNA delivery in cell-laden hydrogels.

mRNA therapeutics have increased in popularity, largely due to the transient and fast nature of protein expression and the low risk of off-target effects. This has increased drastically with the remarkable success of mRNA-based vaccines for COVID-19. Despite advances in lipid nanoparticle (LNP)-based delivery, the mechanisms that regulate efficient endocytic trafficking and translation of mRNA remain poorly understood. Although it is widely acknowledged that the extracellular matrix (ECM) regulates uptake and expression of exogenous nano-complexed genetic material, its specific effects on mRNA delivery and expression have not yet been examined. Here, we demonstrate a critical role for matrix stiffness in modulating both mRNA transfection and expression and uncover distinct mechano-regulatory mechanisms for endocytosis of mRNA through RhoA mediated mTOR signaling and cytoskeletal dynamics. Our findings have implications for effective delivery of therapeutic mRNA to targeted tissues that may be differentially affected by tissue and matrix stiffness.

Impact of atrial fibrillation on the outcomes of transcatheter mitral valve repair using MitraClip: a systematic review and meta-analysis.

Atrial fibrillation (AF) is a common arrhythmia in patients with mitral regurgitation (MR) undergoing transcatheter mitral valve repair (TMVR). In this systematic review, we aimed to investigate the outcomes of TMVR using MitraClip in AF patients. We performed a systematic search using PubMed, SCOPUS, EMBASE, and Google Scholar, from inception to May 10, 2020, for studies that reported outcomes following MitraClip, in patients with AF versus without AF. Seven studies with a total of 7678 patients met the inclusion criteria. The risk of 1-year all-cause mortality following TMVR was higher in AF patients (RR 1.40, 95% CI 1.27-1.54, p ≤ 0.001). Similarly, the risk of heart failure hospitalization was higher in patients with AF (RR 1.17, 95% CI 1.06-1.30, p = 0.002) and the risk of bleeding was elevated in AF patients (RR 1.29, 95% CI 1.15-1.45, p ≤ 0.001). The risk of procedural failure, in-hospital mortality, cardiovascular mortality, and stroke was not significantly different between the two groups. The higher risk of all-cause mortality, HF hospitalization, and risk of bleeding in AF patients undergoing MitraClip warrants attention.

Case Report: Atypical Presentation of Neuroretinitis in a Middle-aged Woman with Pre-existing Maculopathy.

SIGNIFICANCE: Neuroretinitis classically presents with sudden vision loss, disc edema, and macular star formation. However, the classical triad may not always be seen, especially in the case of a pre-existing macular disorder. A thorough clinical examination may still clinch the appropriate diagnosis and prevent unwarranted treatment. PURPOSE: This study aimed to report a case of neuroretinitis in an adult woman with pre-existing type 2 macular telangiectasia where the classical pattern of exudation could not be seen. CASE REPORT: A 48-year-old woman, type 2 diabetic and hypertensive, presented with sudden painless blurring of vision in the right eye for the past 1 week. Macula in both eyes had loss of transparency and intraretinal crystalline deposits and pigments. The right eye had hyperemic edematous disc with peripapillary retinal hemorrhages and hard exudates. Color vision was grossly abnormal, and a centrocecal scotoma was noted on visual field analysis in the right eye. After a normal systemic evaluation, a diagnosis of right eye neuroretinitis and coexistent bilateral type 2 macular telangiectasia (nonproliferative type) was made. Unilateral painless visual loss, severe dyschromatopsia, and peripapillary hard exudates pointed to the correct diagnosis of neuroretinitis. Observation was advised, and visual acuity improved spontaneously. The disc edema resolved with consequent optic atrophy. CONCLUSIONS: Absence of the classical "macular star" appearance does not refute the diagnosis of neuroretinitis when pre-existing maculopathy is present.

Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery.

Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.

Cardiovascular risk reduction with icosapent ethyl.

PURPOSE OF REVIEW: Residual risk for atherosclerotic cardiovascular disease (ASCVD) persists even among patients with optimal low-density lipoprotein cholesterol (LDL-C) levels. Randomized trials attempting to modulate other lipids beyond LDL-C have failed to demonstrate significant reductions in ischemic events. RECENT FINDINGS: Mounting evidence suggests that triglyceride elevation is an independent risk factor for ASCVD. Though trials of triglyceride-lowering therapy in the statin era have failed to provide protection from ASCVD events, subgroup analyses have revealed that those with the highest triglycerides at time of enrollment appeared to receive the greatest clinical benefit. REDUCE-IT was a trial that enrolled patients with high triglycerides despite having goal LDL-C levels on statin therapy. Treatment with icosapent ethyl, a highly purified omega-3 fatty acid (OM3FA), eicosapentaenoic acid ethyl ester, provided a 25% relative risk reduction for the primary composite cardiovascular endpoint (hazard ratio 0.75, 95% CI 0.68--0.83; P = 0.00000001), as well as a 30% relative risk reduction in total ischemic events (P = 0.00000000036). SUMMARY: Icosapent ethyl was rigorously shown to decrease residual risk for cardiovascular events, though the benefits seen were likely because of mechanisms beyond mere triglyceride lowering. Clinical application of icosapent ethyl in this cohort of patients with residual risk is urgently needed.

Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis.

The promise of gene therapy for the treatment of cystic fibrosis has yet to be fully clinically realized despite years of effort toward correcting the underlying genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR). mRNA therapy via nanoparticle delivery represents a powerful technology for the transfer of genetic material to cells with large, widespread populations, such as airway epithelia. We deployed a clinically relevant lipid-based nanoparticle (LNP) for packaging and delivery of large chemically modified CFTR mRNA (cmCFTR) to patient-derived bronchial epithelial cells, resulting in an increase in membrane-localized CFTR and rescue of its primary function as a chloride channel. Furthermore, nasal application of LNP-cmCFTR restored CFTR-mediated chloride secretion to conductive airway epithelia in CFTR knockout mice for at least 14 days. On day 3 post-transfection, CFTR activity peaked, recovering up to 55% of the net chloride efflux characteristic of healthy mice. This magnitude of response is superior to liposomal CFTR DNA delivery and is comparable with outcomes observed in the currently approved drug ivacaftor. LNP-cmRNA-based systems represent a powerful platform technology for correction of cystic fibrosis and other monogenic disorders.

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA.

Intracellular delivery of mRNA holds great potential for vaccine1-3 and therapeutic4 discovery and development. Despite increasing recognition of the utility of lipid-based nanoparticles (LNPs) for intracellular delivery of mRNA, particle engineering is hindered by insufficient understanding of endosomal escape, which is believed to be a main limiter of cytosolic availability and activity of the nucleic acid inside the cell. Using a series of CRISPR-based genetic perturbations of the lysosomal pathway, we have identified that late endosome/lysosome (LE/Ly) formation is essential for functional delivery of exogenously presented mRNA. Lysosomes provide a spatiotemporal hub to orchestrate mTOR signaling and are known to control cell proliferation, nutrient sensing, ribosomal biogenesis, and mRNA translation. Through modulation of the mTOR pathway we were able to enhance or inhibit LNP-mediated mRNA delivery. To further boost intracellular delivery of mRNA, we screened 212 bioactive lipid-like molecules that are either enriched in vesicular compartments or modulate cell signaling. Surprisingly, we have discovered that leukotriene-antagonists, clinically approved for treatment of asthma and other lung diseases, enhance intracellular mRNA delivery in vitro (over 3-fold, p < 0.005) and in vivo (over 2-fold, p < 0.005). Understanding LNP-mediated intracellular delivery will inspire the next generation of RNA therapeutics that have high potency and limited toxicity.

Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations.

Long-term outcomes with first- vs. second-generation drug-eluting stents in saphenous vein graft lesions.

BACKGROUND: As compared with bare metal stents, first-generation drug-eluting stents (DES) improved post-procedural outcomes in aortocoronary saphenous vein graft (SVG) lesions, but there is limited information on outcomes after use of second-generation DES in SVGs. METHODS: We compared the outcomes of patients who received first- (n = 81) with those who received second-generation (n = 166) DES in SVG lesions at our institution between 2006 and 2013. Major adverse cardiac events (MACE) were defined as the composite of all-cause death, myocardial infarction, and target vessel revascularization. RESULTS: Mean age was 66.0 ± 8.1 years and 97.6% of the patients were men. Mean SVG age was 11.1 ± 0.4 years. First-generation DES were sirolimus-eluting (n = 17) and paclitaxel-eluting (n = 64) stents. Second-generation DES were everolimus-eluting (n = 115) and zotarolimus-eluting (n = 51) stents. Median follow-up was 41 months. At 2-years post-procedure, patients with first- and second-generation DES had similar rates of death (20.91% vs. 20.27%, P = 0.916), target lesion revascularization (16.39% vs. 20.00%, P = 0.572), target vessel revascularization (20.97% vs. 23.16%, P = 0.747), myocardial infarction (26.15% vs. 23.00%, P = 0.644), and MACE (43.5% vs. 40.87%, P = 0.707), respectively. CONCLUSIONS: Outcomes with first- and second-generation DES in SVGs are similar. Novel stent designs are needed to further improve the clinical outcomes in this challenging patient and lesion subgroup. © 2015 Wiley Periodicals, Inc.

Transradial approach for coronary chronic total occlusion interventions: Insights from a contemporary multicenter registry.

OBJECTIVES: To examine the impact of transradial access on the procedural outcomes of chronic total occlusion (CTO) percutaneous coronary interventions (PCI). BACKGROUND: The efficacy and safety of transradial access in CTO PCI has received limited study. METHODS: We compared the technique and outcomes of transradial vs. transfemoral access among 650 CTO PCI cases performed between January 2012 and March 2014 at 6 US centers. RESULTS: Most patients were men (87%) with high frequency of diabetes mellitus (42%) and prior coronary artery bypass graft surgery (36%). The CTO target vessel was the right coronary (59%), left anterior descending (20%), or circumflex (17%) artery. TR access was used in 110 (17%) of the 650 cases, as follows: bilateral radial access (63%); bilateral radial access plus unilateral or bilateral femoral access (7%); unilateral radial access plus unilateral or bilateral femoral access (26%); and unilateral radial access (4%). Six and eight French guide catheters were used through the radial and femoral artery, respectively. Compared to transfemoral, transradial cases had similar technical (92.6% vs. 93.0%, P = 0.87) and procedural (91.1% vs. 90.0%, P = 0.95) success and major complication rates (1.7% vs 1.8%, P = 0.99). However, transradial access was associated with higher mean procedure (142 ± 83 vs. 120 ± 60 min, P = 0.008) and fluoroscopy (58 ± 40 vs. 49 ± 31 min, P <0.026) time, and number of crossing approach changes (0.7 ± 1.0 vs. 0.5 ± 0.7, P = 0.008). CONCLUSION: Transradial CTO PCI can be performed with similar success and complication rates with transfemoral CTO PCI, but is associated with longer procedural and fluoroscopy times. © 2015 Wiley Periodicals, Inc.

Depth: a web server to compute depth, cavity sizes, detect potential small-molecule ligand-binding cavities and predict the pKa of ionizable residues in proteins.

Residue depth accurately measures burial and parameterizes local protein environment. Depth is the distance of any atom/residue to the closest bulk water. We consider the non-bulk waters to occupy cavities, whose volumes are determined using a Voronoi procedure. Our estimation of cavity sizes is statistically superior to estimates made by CASTp and VOIDOO, and on par with McVol over a data set of 40 cavities. Our calculated cavity volumes correlated best with the experimentally determined destabilization of 34 mutants from five proteins. Some of the cavities identified are capable of binding small molecule ligands. In this study, we have enhanced our depth-based predictions of binding sites by including evolutionary information. We have demonstrated that on a database (LigASite) of ∼200 proteins, we perform on par with ConCavity and better than MetaPocket 2.0. Our predictions, while less sensitive, are more specific and precise. Finally, we use depth (and other features) to predict pKas of GLU, ASP, LYS and HIS residues. Our results produce an average error of just <1 pH unit over 60 predictions. Our simple empirical method is statistically on par with two and superior to three other methods while inferior to only one. The DEPTH server (http://mspc.bii.a-star.edu.sg/depth/) is an ideal tool for rapid yet accurate structural analyses of protein structures.