OpenFold: retraining AlphaFold2 yields new insights into its learning mechanisms and capacity for generalization.

AlphaFold2 revolutionized structural biology with the ability to predict protein structures with exceptionally high accuracy. Its implementation, however, lacks the code and data required to train new models. These are necessary to (1) tackle new tasks, like protein-ligand complex structure prediction, (2) investigate the process by which the model learns and (3) assess the model's capacity to generalize to unseen regions of fold space. Here we report OpenFold, a fast, memory efficient and trainable implementation of AlphaFold2. We train OpenFold from scratch, matching the accuracy of AlphaFold2. Having established parity, we find that OpenFold is remarkably robust at generalizing even when the size and diversity of its training set is deliberately limited, including near-complete elisions of classes of secondary structure elements. By analyzing intermediate structures produced during training, we also gain insights into the hierarchical manner in which OpenFold learns to fold. In sum, our studies demonstrate the power and utility of OpenFold, which we believe will prove to be a crucial resource for the protein modeling community.

One-Pot Synthesis of Polycyclic 4,5-Dihydropyridazine-3(2H)-ones by Inverse Electron-Demand Diels-Alder (IEDDA) Reactions from Alkenes.

In the classical Inverse Electron-Demand Diels-Alder (IEDDA) reactions between alkenes and tetrazines, 4,5-dihydropyridazines are formed. 4,5-Dihydropyridazines are rapidly converted to the more energetically stable 1,4-dihydropyridazines by 1,3-prototropic isomerization. In this study, instead of 1,4-dihydropyridazines, 4,5-dihydropyridazine-3(2H)-ones were obtained as a result of IEDDA reactions between tetrazines with leaving groups at the 3,6-positions, and norbornene and barrelene-derived polycyclic alkenes in the presence of moisture in air or solvent. To show that this new method works not only on strained polycyclic alkenes but also on monocyclic and linear alkenes, the corresponding 4,5-dihydropyridazine-3(2H)-ones were obtained in high yields from the reactions performed with styrene and cyclopentene as well. The chemical structures of the polycyclic 4,5-dihydropyridazine-3(2H)-ones were determined by NMR and HRMS analyses. In addition, the exact structures of the polycyclic 4,5-dihydropyridazine-3(2H)-ones were also experimentally proven by converting them to pyridazine-3(2H)-ones known in the literature.

From Selection to Instruction and Back: Competing Conformational Selection and Induced Fit Pathways in Abiotic Hosts.

Two limiting cases of molecular recognition, induced fit (IF) and conformational selection (CS), play a central role in allosteric regulation of natural systems. The IF paradigm states that a substrate "instructs" the host to change its shape after complexation, while CS asserts that a guest "selects" the optimal fit from an ensemble of preexisting host conformations. With no studies that quantitatively address the interplay of two limiting pathways in abiotic systems, we herein and for the first time describe the way by which twisted capsule M-1, encompassing two conformers M-1(+) and M-1(-), trap CX4 (X=Cl, Br) to give CX4 ⊂M-1(+) and CX4 ⊂M-1(-), with all four states being in thermal equilibrium. With the assistance of 2D EXSY, we found that CBr4 would, at its lower concentrations, bind M-1 via a M-1(+)→M-1(-)→CBr4 ⊂M-1(-) pathway corresponding to conformational selection. For M-1 complexing CCl4 though, data from 2D EXSY measurements and 1D NMR line-shape analysis suggested that lower CCl4 concentrations would favor CS while the IF pathway prevailed at higher proportions of the guest. Since CS and IF are not mutually exclusive, we reason that our work sets the stage for characterizing the dynamics of a wide range of already existing hosts to broaden our fundamental understanding of their action. The objective is to master the way in which encapsulation takes place for designing novel and allosteric sequestering agents, catalysts and chemosensors akin to those found in nature.

Multivalent C-H⋠⋠⋠Cl/Br-C Interactions Directing the Resolution of Dynamic and Twisted Capsules.

In this work, we report a mechanism by which stereoisomeric and twisted capsules P/M-1 direct their dynamic chirality in the presence of haloalkane guests. The capsule comprises a static, but twisted, cage that is linked to a dynamic tris(2-pyridylmethyl)amine (TPA) lid at its top. From the results of experimental (NMR spectroscopy and X-ray crystallography) and computational (DFT) studies, the TPA lid was shown to assume clockwise (+) and counterclockwise (-) folds with diastereomeric (but racemic) capsules M-1(+) and M-1(-) interconverting at a rapid rate (ΔG≠ 189K =9.1 kcal mol-1 ). The relative stability of the capsules was found to be a function of guest(s) residing in their interior (243/262 Å3 ) with small CH2 Cl2 (61 Å3 ) yielding roughly equal population of diastereomeric inclusion complexes. Larger guests, such as CCl4 (89 Å3 ) and CBr4 (108 Å3 ), however, formed M-1(-)⊂CX4 at the expense of M-1(+)⊂CX4 in circa 3:1 ratio. To account for the observation, theory (DFT:M06-2X/6-31+G*) and experiments (1 H NMR spectroscopy) were used to deduce that CX4 guests become localized inside the twisted cage of the capsule by forming a C-X⋅⋅⋅π halogen bond [Nc =d/(rH +rX )=0.91-0.92] with the benzene "floor" while encountering electrostatic repulsions with closer naphthalimide boundaries. At last, the TPA lid used its central methylene hydrogens to establish, within the M-1(-)⊂CX4 , three stabilizing C-H⋅⋅⋅X-C interactions with the guest. The same C-H⋅⋅⋅X-C interactions, however, became weaker (or possibly vanished) after the conformational reorganization of the lid and the formation of less stable M-1(+)⊂CX4 complex. On individual basis, C-H⋅⋅⋅X-C intermolecular contacts are weak and hardly detectable in the solution phase. In the case of capsule P/M-1, however, these contacts were multivalent and altogether strong enough to direct the host's dynamic chirality.

Inhibition of acetylcholinesterase and butyrylcholinesterase with uracil derivatives: kinetic and computational studies.

Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) inhibitors are interesting compounds for different therapeutic applications, among which Alzheimer's disease. Here, we investigated the inhibition of these cholinesterases with uracil derivatives. The mechanism of inhibition of these enzymes was observed to be due to obstruction of the active site entrance by the inhibitors scaffold. Molecular docking and molecular dynamics (MD) simulations demonstrated the possible key interactions between the studied ligands and amino acid residues at different regions of the active sites of AChE and BuChE. Being diverse of the classical AChE and BuChE inhibitors, the investigated uracil derivatives may be used as lead molecules for designing new therapeutically effective enzyme inhibitors.

Effects of aryl methanesulfonate derivatives on acetylcholinesterase and butyrylcholinesterase.

There is a dire need for new treatments for Alzheimer's disease (AD). Principal drugs have reached maturity, and the number of people affected by AD is growing at a rapid rate. After years of research and many clinical trials, only symptomatic treatments are available. An effective disease-modifying drug for AD needs to be discovered. The research presented in this paper aims to facilitate in the discovery of new potential targets that could help in the ongoing AD research. Aryl methanesulfonate derivatives were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. IC50 values between 0.660 and 3.397 µM against AChE and 0.885 and 2.596 µM against BuChE were obtained.

Characterization of hawthorn (Crataegus spp.) genotypes by SSR markers.

Hawthorn (Crataegus spp.) is an edible wild fruit that is used in traditional medicine, landscape studies, and food and beverage industries in many countries. It is an important wild plant species in Turkey and is numerous in the Yozgat Province. Genetic and breeding studies on hawthorn are very limited. Therefore, we aimed to characterize 91 hawthorn genotypes using simple sequence repeat (SSR) markers. The SSRs were developed from apple and pear and were screened in hawthorn for amplification and polymorphisms. A total of 265 alleles were detected from thirty-two SSR primer pairs, and those were used to identify genetic relationships. The number of alleles ranged from 2 to 21 alleles per locus with a mean value of 8.28. The Hi05b09 locus showed the highest allele number (Na = 21). The polymorphism information content (PIC) values ranged from 0.16 (CH03d10) to 0.89 (C6554) with a mean value of 0.60. An Unweighted Pair Group Method with Arithmetic Average method was used to cluster the genotypes, and four major clusters were obtained from the amplification of the SSRs. STRUCTURE software identified four populations (ΔK = 4) and eight sub-populations (ΔK = 8), and four major clusters similar results to UPGMA analysis. Our study showed that the SSR markers could be utilized as a reliable tool for the determination of genetic variations and relationships of hawthorn genotypes. A basic molecular analysis on the hawthorn genotypes identified in this study will promote the collection of germplasm collection and the selection of parents' in future cross-breeding studies.

Coexistence of Gitelman Syndrome and Hypertrophic Cardiomyopathy in a Pregnant Woman.

Gitelman syndrome (GS) is transmitted as an autosomal recessive trait and characterized by hypokalemic metabolic alkalosis in combination with significant hypomagnesemia and low urinary calcium excretion. The symptoms and severity of the disease can vary greatly from one person to another and can range from mild to severe. Sudden cardiac arrest has been reported occasionally as well. Here, for the first time, we reported a 34-year-old pregnant GS woman who was diagnosed to have hypertrophic obstructive cardiomyopathy during her cardiac examination for the complaints of palpitation and presyncope.

Coronary Artery Dominance May Predict Future Risk of Atrial Fibrillation.

BACKGROUND: Ischemia of the atria and conductive system of the heart results in greater atrial electrophysiological changes and propensity for atrial fibrillation. P wave duration and dispersion have been proposed to be useful for the prediction of paroxysmal atrial fibrillation (PAF). This study aimed to investigate the effect of coronary artery dominance on P wave duration and dispersion. METHODS: The study population included 194 patients with left dominant circulation (LDC) and 200 age- and gender-matched controls with right dominant circulation (RDC) and without coronary artery disease based on invasive coronary angiography findings. P wave dispersion (PWD) was defined as the difference between the maximum and minimum P wave duration. Arrhythmias were identified by 24-hour Holter electrocardiogram at 3 years of follow-up. RESULTS: PWD was significantly prolonged in the patients with LDC compared to the controls with RDC (p = 0.001). There were positive correlations between PWD and age (r: 0.502, p = 0.009), left ventricular mass (LVM) (r: 0.614, p = 0.001), LVM index (r: 0.727, p < 0.001) and left atrium (LA) diameter (r: 0.558, p = 0.003) in the LDC group. Multivariate logistic regression analysis showed that age, LVM index, LA diameter and LDC were independent predictors of prolonged PWD. At 3 years of follow-up, 7 (3.9%) patients with LDC and 1 (0.5%) patient with RDC had PAF in Holter electrocardiogram (p < 0.001). CONCLUSIONS: LDC could lead to an increased risk of atrial fibrillation through prolonged PWD. We recommend following up these patients to assess the development of atrial fibrillation.

Quadricuspid Aortic Valve Associated with Aortic Regurgitation, Mitral Regurgitation, and Aortic Dilatation.

The quadricuspid aortic valve (QAV) is a very uncommon congenital malformation with an estimated incidence of 0.003% to 0.043% of all congenital heart diseases. Combinations of QAV with several different congenital malformations have been described. The case is reported of a type A QAV associated with moderate aortic regurgitation, mild mitral regurgitation, and ascending aorta dilatation. This interesting case was referred for close follow up.

An unusual presentation of papillary fibroelastoma originating from right ventricular outflow tract.

Papillary fibroelastomas (PFEs) are primary cardiac tumors with a benign and avascular nature. Majority of the PFEs are originated from the valvular endocardium, while the most common site is aortic valve. In this case, we present a patient with multiple PFEs originating from the right ventricular outflow tract who was admitted to our clinic with exertional dyspnea. As far as we know, this is the first case of this unusual presentation of multiple PFEs and also had a history of breast cancer and permanent pacemaker reported in the literature.

Identification of sex-linked SNP markers using RAD sequencing suggests ZW/ZZ sex determination in Pistacia vera L.

BACKGROUND: Pistachio (Pistacia vera L.) is a dioecious species that has a long juvenility period. Therefore, development of marker-assisted selection (MAS) techniques would greatly facilitate pistachio cultivar-breeding programs. The sex determination mechanism is presently unknown in pistachio. The generation of sex-linked markers is likely to reduce time, labor, and costs associated with breeding programs, and will help to clarify the sex determination system in pistachio. RESULTS: Restriction site-associated DNA (RAD) markers were used to identify sex-linked markers and to elucidate the sex determination system in pistachio. Eight male and eight female F1 progenies from a Pistacia vera L. Siirt × Bagyolu cross, along with the parents, were subjected to RAD sequencing in two lanes of a Hi-Seq 2000 sequencing platform. This generated 449 million reads, comprising approximately 37.7 Gb of sequences. There were 33,757 polymorphic single nucleotide polymorphism (SNP) loci between the parents. Thirty-eight of these, from 28 RAD reads, were detected as putative sex-associated loci in pistachio. Validation was performed by SNaPshot analysis in 42 mature F1 progenies and in 124 cultivars and genotypes in a germplasm collection. Eight loci could distinguish sex with 100% accuracy in pistachio. To ascertain cost-effective application of markers in a breeding program, high-resolution melting (HRM) analysis was performed; four markers were found to perfectly separate sexes in pistachio. Because of the female heterogamety in all candidate SNP loci, we report for the first time that pistachio has a ZZ/ZW sex determination system. As the reported female-to-male segregation ratio is 1:1 in all known segregating populations and there is no previous report of super-female genotypes or female heteromorphic chromosomes in pistachio, it appears that the WW genotype is not viable. CONCLUSION: Sex-linked SNP markers were identified and validated in a large germplasm and proved their suitability for MAS in pistachio. HRM analysis successfully validated the sex-linked markers for MAS. For the first time in dioecious pistachio, a female heterogamety ZW/ZZ sex determination system is suggested.

Synthesis and carbonic anhydrase inhibitory properties of novel uracil derivatives.

Carbonic anhydrase (CA) inhibitors are valuable molecules based on several therapeutic applications, including antiglaucoma activity. In the present study, inhibition of two human cytosolic carbonic anhydrase isozymes I and II with some uracil derivatives (3-9) were investigated. Compounds 3-9 showed KI values in the range of 10.83-464 µM for hCA I and of 28.88-778.5 µM against hCA II, respectively. Kinetic investigations showed that similarly to classical CA inhibitors, all investigated natural compounds act as competitive inhibitors with 4-NPA as substrate. Uracil derivatives investigated here are promising agents which may be used as lead molecules in order to derivative novel carbonic anhydrase inhibitors that might be useful in medical applications.

Comparison of lipids, fatty acids and volatile compounds of various kumquat species using HS/GC/MS/FID techniques.

BACKGROUND: Kumquat (Fortunella spp.) is one of the important fruit species that has been introduced into Turkey recently. It is well adapted to the Mediterranean region in Turkey. Early research of kumquat was started on adaptation and pomological traits, and only a few studies have been concerned with chemical content. After understanding the health benefits of kumquat fruits farmers started their plantations in Turkey. Thus in this study the fruits of five kumquat species - Fortunella margarita (Lour.) Swingle, F. crassifolia Swingle, F. obovata Hort. ex Tanaka, F. hindsii (Champ. ex Benth.) Swingle, and limequat [Citrus aurantifolia × F. japonica (Thumb.)] - were compared based on their lipids, fatty acids and volatile profiles. The lipids, fatty acids and volatile profiles were determined by using gas chromatography with flame ionisation detection (GC/FID) and headspace gas chromatography mass spectrometry (HS-GC/MS) techniques. Volatile constituents of kumquat fruits were isolated by a headspace method. RESULTS: The total lipid content (%) of kumquat species ranged from 0.26% to 0.37%. The polyunsaturated fatty acids were detected in a higher amount compared to the monounsaturated fatty acids and saturated fatty acids, respectively. The headspace technique led to the identification of 39 compounds and among a total of 39 constituents identified, d-limonene was the most abundant compound, comprising 67.78-88.72%. The results indicated that kumquats are rich in dietary and health benefits. CONCLUSION: Thirty-nine compounds were identified and terpenes were the major chemical group in all kumquat genotypes. Esters, alcohols, aldehydes and ketones were also detected. The present study revealed that the kumquat fruits have a valuable levels of health beneficial chemo-preventive effects and genotypes varied based on their lipid, fatty acids and volatile constituents.

Oxidation of cyanobenzocycloheptatrienes: Synthesis, photooxygenation reaction and carbonic anhydrase isoenzymes inhibition properties of some new benzotropone derivatives.

The oxidation of some cyanocycloheptatrienes with CrO3 and pyridine was investigated and a few new nitrile functionalised benzotropone derivatives were obtained. Photooxygenation reaction of these products was also studied. The structures of the formed products were determined on the basis of NMR spectroscopy and the formation mechanism of unusual products was discussed. Human carbonic anhydrase isoenzymes I, and II (hCA I and hCA II) inhibition properties of nitrile functionalized new benzotropone derivatives were also studied. Both CA isozymes were inhibited in the low micromolar range by these nitrile functionalized benzotropone analogues. The newly synthesized benzotropone derivatives showed inhibition constants in the sub-micromolar range (2.51-4.06µM). The best hCA I inhibition was observed in 5H-benzocycloheptene-7-carbonitrile (Ki: 2.88±0.86µM). On the other hand, 5-oxo-5H-benzocycloheptatriene-7-carbonitrile showed the powerful inhibitory effect against hCA II (Ki: 2.51±0.34µM).

Nucleophilic substitution of bromonorbornenes and derivatives by electron transfer reactions.

The photoinitiated substitution reactions of anti-7-bromobenzonorbornadiene (5), its syn isomer 6, exo-anti-13-bromobenzocyclobutanorbornene (7), syn-7-bromonorbornene (8) and bromonorbornane (9) with Me(3)Sn(-) and Ph(2)P(-) anions, in liquid ammonia, are here informed to occur with good yields of substitution. The stereochemical outcome is discussed in terms of calculations with the B3LYP functional and the 6-31+G* basis set; the solvent being included as a continuum through the PCM model. The experimental relative chemical reactivity of pairs of substrates toward a given anion is also presented.

Transcatheter Aortic Valve Implantation in Bicuspid Aortic Valve Patients with Coagulation Factor 7 and 11 Deficiency and Atrial Fibrillation.

Although transcatheter aortic valve implantation (TAVI), which is a less invasive standard treatment for aortic stenosis than surgery, has been recommended even in low-risk patients, its effectiveness in bicuspid aortic valve is still unclear. Cardiac surgery has been proven to cause serious complications in hematological diseases with factor deficiency or bleeding diathesis. In this case, which is the first in the literature to our knowledge, we tried to present the successful TAVI procedure in a young patient with bicuspid aortic stenosis and factor 7, 11 deficiency complicated by atrial fibrillation.

Pistachio genomes provide insights into nut tree domestication and ZW sex chromosome evolution.

Pistachio is a nut crop domesticated in the Fertile Crescent and a dioecious species with ZW sex chromosomes. We sequenced the genomes of Pistacia vera cultivar (cv.) Siirt, the female parent, and P. vera cv. Bagyolu, the male parent. Two chromosome-level reference genomes of pistachio were generated, and Z and W chromosomes were assembled. The ZW chromosomes originated from an autosome following the first inversion, which occurred approximately 8.18 Mya. Three inversion events in the W chromosome led to the formation of a 12.7-Mb (22.8% of the W chromosome) non-recombining region. These W-specific sequences contain several genes of interest that may have played a pivotal role in sex determination and contributed to the initiation and evolution of a ZW sex chromosome system in pistachio. The W-specific genes, including defA, defA-like, DYT1, two PTEN1, and two tandem duplications of six VPS13A paralogs, are strong candidates for sex determination or differentiation. Demographic history analysis of resequenced genomes suggest that cultivated pistachio underwent severe domestication bottlenecks approximately 7640 years ago, dating the domestication event close to the archeological record of pistachio domestication in Iran. We identified 390, 211, and 290 potential selective sweeps in 3 cultivar subgroups that underlie agronomic traits such as nut development and quality, grafting success, flowering time shift, and drought tolerance. These findings have improved our understanding of the genomic basis of sex determination/differentiation and horticulturally important traits and will accelerate the improvement of pistachio cultivars and rootstocks.

Simple methanesulfonates are hydrolyzed by the sulfatase carbonic anhydrase activity.

The possible sulfatase activity of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms have been investigated with a series of synthesized methanesulfonate derivatives of phenols. Four α-CA isozymes, i.e. hCA I, hCA II, hCA IV and hCA VI (h = human isoform), were included in the study. We evidenced that the original sulfonate esters are being hydrolyzed effectively to the corresponding phenols which there after act as CA inhibitors. The K(I)-s of these compounds ranged from 10.24 to 4012 µM against hCA I, 0.10 to 35.42 µM against hCA II, 0.49 to 45.06 µM against hCA IV and 3.27 to 608 µM against CA VI, respectively. The relevant sulfatase activity of CA with these esters is amazing considering the fact that 4-nitrophenyl-sulfate, an activated ester, is not a substrate of these enzymes.

Predictors and Prognostic Implications of Myocardial Injury After Transcatheter Aortic Valve Replacement.

Myocardial injury (MI) is not unusual after transcatheter aortic valve replacement (TAVR). To determine precipitating factors and prognostic outcomes of MI after TAVR, we retrospectively investigated relationships between MI after TAVR and aortic root dimensions, baseline patient characteristics, echocardiographic findings, and procedural features. Of 474 patients who underwent transfemoral TAVR for severe aortic stenosis in our tertiary center from June 2011 through June 2018, 188 (mean age, 77.7 ± 7.7 yr; 96 women [51%]) met the study inclusion criteria. Patients were divided into postprocedural MI (PMI) (n=74) and no-PMI (n=114) groups, in accordance with high-sensitivity troponin T levels. We found that MI risk was associated with older age (odds ratio [OR]=1.054; 95% CI, 1.013-1.098; P=0.01), transcatheter heart valve type (OR=10.207; 95% CI, 2.861-36.463; P=0.001), distances from the aortic annulus to the right coronary artery ostium (OR=0.853; 95% CI, 0.731-0.995; P=0.04) and the left main coronary artery ostium (OR=0.747; 95% CI, 0.616-0.906; P=0.003), and baseline glomerular filtration rate (OR=0.985; 95% CI, 0.970-1.000; P=0.04). Moreover, the PMI group had a longer time to hospital discharge (P=0.001) and a higher permanent pacemaker implantation rate (P=0.04) than did the no-PMI group. Our findings may enable better estimation of which patients are at higher risk of MI after TAVR and thus improve the planning and course of clinical care.