Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ∼1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

Long-term outcomes of hospitalized patients with SARS-CoV-2/COVID-19 with and without neurological involvement: 3-year follow-up assessment.

BACKGROUND: Acute neurological manifestation is a common complication of acute Coronavirus Disease 2019 (COVID-19) disease. This retrospective cohort study investigated the 3-year outcomes of patients with and without significant neurological manifestations during initial COVID-19 hospitalization. METHODS AND FINDINGS: Patients hospitalized for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection between 03/01/2020 and 4/16/2020 in the Montefiore Health System in the Bronx, an epicenter of the early pandemic, were included. Follow-up data was captured up to 01/23/2023 (3 years post-COVID-19). This cohort consisted of 414 patients with COVID-19 with significant neurological manifestations and 1,199 propensity-matched patients (for age and COVID-19 severity score) with COVID-19 without neurological manifestations. Neurological involvement during the acute phase included acute stroke, new or recrudescent seizures, anatomic brain lesions, presence of altered mentation with evidence for impaired cognition or arousal, and neuro-COVID-19 complex (headache, anosmia, ageusia, chemesthesis, vertigo, presyncope, paresthesias, cranial nerve abnormalities, ataxia, dysautonomia, and skeletal muscle injury with normal orientation and arousal signs). There were no significant group differences in female sex composition (44.93% versus 48.21%, p = 0.249), ICU and IMV status, white, not Hispanic (6.52% versus 7.84%, p = 0.380), and Hispanic (33.57% versus 38.20%, p = 0.093), except black non-Hispanic (42.51% versus 36.03%, p = 0.019). Primary outcomes were mortality, stroke, heart attack, major adverse cardiovascular events (MACE), reinfection, and hospital readmission post-discharge. Secondary outcomes were neuroimaging findings (hemorrhage, active and prior stroke, mass effect, microhemorrhages, white matter changes, microvascular disease (MVD), and volume loss). More patients in the neurological cohort were discharged to acute rehabilitation (10.39% versus 3.34%, p < 0.001) or skilled nursing facilities (35.75% versus 25.35%, p < 0.001) and fewer to home (50.24% versus 66.64%, p < 0.001) than matched controls. Incidence of readmission for any reason (65.70% versus 60.72%, p = 0.036), stroke (6.28% versus 2.34%, p < 0.001), and MACE (20.53% versus 16.51%, p = 0.032) was higher in the neurological cohort post-discharge. Per Kaplan-Meier univariate survival curve analysis, such patients in the neurological cohort were more likely to die post-discharge compared to controls (hazard ratio: 2.346, (95% confidence interval (CI) [1.586, 3.470]; p < 0.001)). Across both cohorts, the major causes of death post-discharge were heart disease (13.79% neurological, 15.38% control), sepsis (8.63%, 17.58%), influenza and pneumonia (13.79%, 9.89%), COVID-19 (10.34%, 7.69%), and acute respiratory distress syndrome (ARDS) (10.34%, 6.59%). Factors associated with mortality after leaving the hospital involved the neurological cohort (odds ratio (OR): 1.802 (95% CI [1.237, 2.608]; p = 0.002)), discharge disposition (OR: 1.508 (95% CI [1.276, 1.775]; p < 0.001)), congestive heart failure (OR: 2.281 (95% CI [1.429, 3.593]; p < 0.001)), higher COVID-19 severity score (OR: 1.177 (95% CI [1.062, 1.304]; p = 0.002)), and older age (OR: 1.027 (95% CI [1.010, 1.044]; p = 0.002)). There were no group differences in radiological findings, except that the neurological cohort showed significantly more age-adjusted brain volume loss (p = 0.045) than controls. The study's patient cohort was limited to patients infected with COVID-19 during the first wave of the pandemic, when hospitals were overburdened, vaccines were not yet available, and treatments were limited. Patient profiles might differ when interrogating subsequent waves. CONCLUSIONS: Patients with COVID-19 with neurological manifestations had worse long-term outcomes compared to matched controls. These findings raise awareness and the need for closer monitoring and timely interventions for patients with COVID-19 with neurological manifestations, as their disease course involving initial neurological manifestations is associated with enhanced morbidity and mortality.

PDK1:PKCα heterodimer association-dissociation dynamics in single-molecule diffusion tracks on a target membrane.

Previous studies have documented the formation of a heterodimer between the two protein kinases PDK1 and PKCα on a lipid bilayer containing their target lipids. This work investigates the association-dissociation kinetics of this PDK1:PKCα heterodimer. The approach monitors the two-dimensional diffusion of single, membrane-associated PDK1 molecules for diffusivity changes as PKCα molecules bind and unbind. In the absence of PKCα, a membrane-associated PDK1 molecule exhibits high diffusivity (or large diffusion constant, D) because its membrane-contacting PH domain binds the target PIP3 lipid headgroup with little bilayer penetration, yielding minimal frictional drag against the bilayer. In contrast, membrane-associated PKCα contacts the bilayer via its C1A, C1B, and C2 domains, which each bind at least one target lipid with significant bilayer insertion, yielding a large frictional drag and low diffusivity. The present findings reveal that individual fluor-PDK1 molecules freely diffusing on the membrane surface undergo reversible switching between distinct high and low diffusivity states, corresponding to the PDK1 monomer and the PDK1:PKCα heterodimer, respectively. The observed single-molecule diffusion trajectories are converted to step length time courses, then subjected to two-state, hidden Markov modeling and dwell time analysis. The findings reveal that both the PDK1 monomer state and the PDK1:PKCα heterodimer state decay via simple exponential kinetics, yielding estimates of rate constants for state switching in both directions. Notably, the PDK1:PKCα heterodimer has been shown to competitively inhibit PDK1 phosphoactivation of AKT1, and is believed to play a tumor suppressor role by limiting excess activation of the highly oncogenic PDK1/AKT1/mTOR pathway. Thus, the present elucidation of the PDK1:PKCα association-dissociation kinetics has important biological and medical implications. More broadly, the findings illustrate the power of single-molecule diffusion measurements to reveal the kinetics of association-dissociation events in membrane signaling reactions that yield a large change in diffusive mobility.

Long-term clinical outcomes and healthcare utilization of sickle cell disease patients with COVID-19: A 2.5-year follow-up study.

OBJECTIVES: This study investigated whether patients with sickle cell disease (SCD) had elevated risk of worse long-term clinical outcomes and healthcare utilization 2.5 years post-SARS-CoV-2 infection. METHODS: This study consisted of 178 patients with SCD who tested positive for COVID-19 between February 1, 2020 and January 30, 2022 in a major academic health system in New York City. The control cohort consisted of two-to-one matches of 356 SCD patients without a COVID-19 positive test. The last follow-up was July 18, 2022. The primary outcome was mortality. Secondary outcomes were annualized emergency department visits due to pain, pain hospital admission, length of stay due to pain, acute chest syndrome, episodic transfusion, and episodic exchange transfusion. RESULTS: There was no significant difference in mortality between SCD patients with and without COVID-19 (p > .05). There were no significant differences in secondary outcomes between pre- and postpandemic (p > .05). There were also no significant differences in these outcomes between SCD patients with and without COVID-19 (p > .05). SCD care utilization was not significantly associated with COVID-19 hospitalization status (p > .05). CONCLUSIONS: SCD patients with SARS-CoV-2 infection incurred no additional risk of worse long-term outcomes compared to matched controls of SCD patients not infected by SARS-CoV-2.

Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes.

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

Single-molecule studies reveal regulatory interactions between master kinases PDK1, AKT1, and PKC.

Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, whereas PKCα is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.

The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism.

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.

Patients with unmet social needs are at higher risks of developing severe long COVID-19 symptoms and neuropsychiatric sequela.

This study investigated long COVID of patients in the Montefiore Health System COVID-19 (CORE) Clinics in the Bronx with an emphasis on identifying health related social needs (HRSNs). We analyzed a cohort of 643 CORE patients (6/26/2020-2/24/2023) and 52,089 non-CORE COVID-19 patients. Outcomes included symptoms, physical, emotional, and cognitive function test scores obtained at least three months post-infection. Socioeconomic variables included median incomes, insurance status, and HRSNs. The CORE cohort was older age (53.38 ± 14.50 vs. 45.91 ± 23.79 years old, p < 0.001), more female (72.47% vs. 56.86%, p < 0.001), had higher prevalence of hypertension (45.88% vs. 23.28%, p < 0.001), diabetes (22.86% vs. 13.83%, p < 0.001), COPD (7.15% vs. 2.28%, p < 0.001), asthma (25.51% vs. 12.66%, p < 0.001), lower incomes (53.81% vs. 43.67%, 1st quintile, p < 0.001), and more unmet social needs (29.81% vs. 18.49%, p < 0.001) compared to non-CORE COVID-19 survivors. CORE patients reported a wide range of severe long-COVID symptoms. CORE patients with unmet HRSNs experienced more severe symptoms, worse ESAS-r scores (tiredness, wellbeing, shortness of breath, and pain), PHQ-9 scores (12.5 (6, 17.75) vs. 7 (2, 12), p < 0.001), and GAD-7 scores (8.5 (3, 15) vs. 4 (0, 9), p < 0.001) compared to CORE patients without. Patients with unmet HRSNs experienced worse long-COVID outcomes compared to those without.

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ~1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 Å to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.

Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co(2+) bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

[Delayed diagnosis of carcinoma of the oral cavity].

Approximately 4% of cancers are located in the mouth and pharynx, 95% of which are squamous cell carcinoma (SCC). The case fatality rate of SCC is very high 50%, but early diagnosis significantly lowers the fatality rate. Diagnosis and treatment of oral lesions are shared by numerous specialists including dermatologists, otolaryngeologists, family physicians, general dentists and specialists in oral medicine and in oral surgery. The article describes a patient who was referred to a dermatologist because of a lesion of two months duration on the lateral aspects of the tongue. The dermatologist clinically diagnosed an apthous ulcer and treated it as such. When he returned after two and a half months after no clinical improvement he was diagnosed as suffering from candidiasis and treated accordingly. The unsatisfied patient turned to another dermatologist who referred him to a specialist in oral surgery. He immediately performed a biopsy. SSC was diagnosed, one third of the tongue was resected as well as the submandibular lymph nodes and salivary gland. An Israeli study has demonstrated that dentists performed better than physicians from general medical specialist fields in diagnosing early stage oral cancer. The authors suggest that patients suffering from oral lesions should be referred to specialists in oral medicine or in oral surgery for the diagnosis of oral lesions in order to diagnose oral cancer earlier.

Fracture resistance of endodontically treated teeth restored with combined composite-amalgam restorations.

OBJECTIVE: To evaluate the resistance to fracture of endodontically treated teeth restored with combined composite-amalgam restorations in comparison to all-amalgam restorations. METHOD AND MATERIALS: Forty-eight human premolar teeth were equally divided into 4 groups. Mesio-occlusodistal (MOD) cavities were prepared in 3 groups, and in the fourth group, a modified MOD preparation was designed with an additional buccolingual groove. All teeth were endodontically treated and restored using 1 of several restorative modalities: all amalgam (AM), all amalgam plus dentin adhesive (ADA), amalgam plus dentin adhesive plus composite resin (ADAC), and amalgam plus dentin adhesive plus composite resin with a modified preparation design (ADACM). Specimens were tested in a universal testing machine (Instron). The load (in kilonewtons) at fracture was recorded and statistically analyzed using a Bonferroni one-way statistical analysis (significance: P

Dento-alveolar and maxillofacial injuries - a survey of knowledge of the regimental aid providers in the Israeli army.

The present study evaluated the knowledge of physicians and emergency medical technicians (EMT) regarding primary treatment for oral and maxillofacial trauma and assessed the experience they have in treating oral and maxillofacial injuries. The study population consisted of 80 military physicians and EMT during their military service. A questionnaire was distributed relating to demographic data such as age, gender, position, and type of military service, as well as past experience in treating or witnessing oral and maxillofacial trauma, former education regarding diagnosis and treatment of oral and maxillofacial trauma, assessment of knowledge regarding oral and maxillofacial trauma, etc. The questionnaire was answered by 76 participants (95% response rate): 32 physicians and 44 EMT. Only 17 (22.4%) received education regarding oral and maxillofacial trauma (eight physicians, 25% and nine EMT, 20.5%). Nevertheless, 23 (30.3%) reported witnessing such an injury during their military service. Oral and maxillofacial injuries were first seen by the EMT in 43.4% of the cases, a physician in 23.7%, and a dentist in only 9.2%. Overall, 66 (86.8%) of the physicians and EMT stated that it was important to educate the primary health care providers regarding diagnosis and treatment of oral and maxillofacial trauma. Special emphasis should be given to providing primary caregivers with the relevant education to improve their knowledge and ability of dealing with diagnosis and treatment of oral and maxillofacial trauma.

Assessment of the resistance to fracture of endodontically treated molars restored with amalgam.

STATEMENT OF PROBLEM: Controversy exists concerning the preferred type of final restoration of endodontically treated molars regarding their resistance to fracture under occlusal load. PURPOSE: The aim of this study was to assess the resistance to fracture of endodontically treated molars with various degrees of tooth structure loss restored with amalgam under simulated occlusal load. MATERIAL AND METHODS: . The study teeth consisted of 90 noncarious, nonrestored molars stored in physiological saline solution. The teeth were endodontically treated and randomly divided into 9 experimental groups of 10 specimens each with various degrees of tooth loss, ranging from a conservative endodontic access to removal of all cusps. All teeth were restored with a standardized technique to their original contour with amalgam. Each specimen was mounted onto a specialized jig for loading at the central fossa at a 30-degree angle to the long tooth axis. The resistance to fracture, under continuous compressive force at a cross-head speed of 2 mm/minute, was applied by a universal testing machine and was recorded. One-way analysis of variance with Scheffé contrast was used to statistically compare the differences between the groups at significance level P<.05. RESULTS: One-way analysis of variance showed that the group with a conservative endodontic access (1137.6 +/- 311.6 N) and the group with removal of all cusps (1261.4 +/- 195.1 N) presented a significantly higher resistance to fracture when compared with the other groups (P<.05). There was no significant difference in resistance to fracture under the simulated load between the other 7 groups, which ranged from 655.8 +/- 229.4 to 906.3 +/- 168.1 N (P>.05). CONCLUSION: Within the limitations of this study, the endodontically treated molars with a conservative endodontic access or after removal of all cusps that were restored to their original contour with amalgam presented the highest resistance to fracture under a simulated occlusal load.