Native State Research Articles

SCIENTIFIC AND METHODOLOGICAL PRODUCTION OF THE DEPARTMENT OF UKRAINIAN APPLIED LINGUISTICS FOR TWENTY YEARS

A review of the scientific and methodological output of the teachers of the Department of Ukrainian Applied Linguistics for two decades was carried out. Established in 1996, the department has become a strategically important structural unit both for the popularization of the Ukrainian language in a foreign language environment and for the significant improvement of the scientific-instrumental level of professional speech of students of other faculties of the university. To ensure successful teaching work, it was necessary to have appropriate educational materials both for teaching the Ukrainian language to foreign students and for students of various professional fields, improving their mastery of the native (state) language and scientific and professional vocabulary. Among the publications of the department are textbooks, manuals, dictionaries, phrasebooks, materials for reading in a foreign-language audience, collections of scientific works, in which acute issues of the methodology of language teaching to foreigners were considered and are being considered, materials on linguistic and regional studies, which included texts on the history of Ukraine, its culture, art, folk traditions. The department became a pioneer in the development of national standards for the Ukrainian language for foreigners, developed a scale of levels of Ukrainian language proficiency, requirements for knowledge, abilities and skills of foreign citizens, which became the basis for both the Standard of the Lviv National University and the State Standard. In lectures and practical classes in a professional direction, the main attention is focused on the Ukrainian language course through the prism of language norms and special terminology in order to bring students closer to their future profession, to produce a literate, competent, highly cultured specialist. Key words: Ukrainian languageas a foreign language, Ukrainian language of specialization, business Ukrainian broad casting, scientific and methodical products of the department.

In-droplet hydrogen-deuterium exchange to examine protein/peptide solution conformer heterogeneity.

Many different structure analysis techniques are not capable of probing the heterogeneity of solution conformations. Here, we examine the ability of in-droplet hydrogen-deuterium exchange (HDX) to directly probe solution conformer heterogeneity of a protein with mass spectrometry (MS) detection. Two vibrating capillary vibrating sharp-edge spray ionization (cVSSI) devices have been arranged such that they generate microdroplet plumes of the analyte and D2 O reagent, which coalesce to form reaction droplets where HDX takes place in the solution environment. The native HDX-MS setup has been first explored for two model peptides that have distinct structural compositions in solution. The effectiveness of the multidevice cVSSI-HDX in illustrating structural details has been further exploited to investigate coexisting solution-phase conformations of the protein ubiquitin. In-droplet HDX reveals decreased backbone exchange for a model peptide that has a greater helix-forming propensity. Differences in intrinsic rates of the alanine and serine residues may account for much of the observed protection. The data allow the first estimates of backbone exchange rates for peptides undergoing in-droplet HDX. That said, the approach may hold greater potential for investigating the tertiary structure and structural transitions of proteins. For ubiquitin protein, HDX reactivity differences suggest that multiple conformers are present in native solutions. The addition of methanol to buffered aqueous solutions of ubiquitin results in increased populations of solution conformers of higher reactivity. Data analysis suggests that partially folded conformers such as the A-state of ubiquitin increase with methanol content; the native state may be preserved to a limited degree even under stronger denaturation conditions. The deuterium uptake after in-droplet HDX has been observed to correspond to some degree with peptide backbone hydrogen protection based on differences in intrinsic rates of exchange. The presence of coexisting protein solution structures under native and denaturing solution conditions has been distinguished by the isotopic distributions of deuterated ubiquitin ions.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have ‘escaped’ the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Biomechanical Evaluation of Posterior Shoulder Instability With a Clinically Relevant Posterior Glenoid Bone Loss Model

Background: Existing biomechanical studies of posterior glenoid bone loss and labral pathology are limited by their use of anterior instability models, which differ in both orientation and morphology and have been performed in only a single, neutral arm position. Purpose: To evaluate the biomechanical effectiveness of a posterior labral repair in the setting of a clinically relevant posterior bone loss model in various at-risk arm positions. Study Design: Controlled laboratory study. Methods: Ten fresh-frozen cadaveric shoulders were tested in 7 consecutive states using a 6 degrees of freedom robotic arm: (1) native, (2) posterior labral tear (6-9 o’clock), (3) posterior labral repair, (4) mean posterior glenoid bone loss (7%) with labral tear, (5) mean posterior glenoid bone loss with labral repair, (6) large posterior glenoid bone loss (28%) with labral tear, and (7) large posterior glenoid bone loss with labral repair. Bone loss was created using 3-dimensional printed computed tomography model templates. Biomechanical testing consisted of 75 N of posterior-inferior force and 75 N of compression at 60° and 90° of flexion and scaption. Posterior-inferior translation, lateral translation, and peak dislocation force were measured for each condition. Results: Labral repair significantly increased dislocation force independent of bone loss state between 10.1 and 14.8 N depending on arm position. Dislocation force significantly decreased between no bone loss and small bone loss (11.9-13.5 N), small bone loss and large bone loss (9.4-14.3 N), and no bone loss and large bone loss (21.2-26.5 N). Labral repair significantly decreased posterior-inferior translation compared with labral tear states by a range of 1.0 to 2.3 mm. In the native state, the shoulder was most unstable in 60° of scaption, with 29.9 ± 6.1-mm posterior-inferior translation. Conclusion: Posterior labral repair improved stability of the glenohumeral joint, and even in smaller to medium amounts of posterior glenoid bone loss the glenohumeral stability was maintained with labral repair in this cadaveric model. However, a labral repair with large bone loss could not improve stability to the native state. Clinical Relevance: This study shows that larger amounts of posterior glenoid bone loss (>25%) may require bony augmentation for adequate stability.

Effect of Choline Amino Acid-Based Ionic Liquids on Stability and Structure of Hemoglobin.

Ionic liquids (ILs) can stabilize or destabilize proteins, which motivates us to examine their effect on hemoglobin. The native state of hemoglobin (Hb) is disrupted at different physical conditions such as pressure, temperature, and solvents. Herein, we have monitored the stability of Hb in a nontoxic and biocompatible IL, i. e., choline amino acid-based Ils (ChAAILs), using various spectroscopic techniques like UV-Vis and fluorescence spectroscopy, circular dichroism (CD), and isothermal titration calorimetry (ITC) measurements. It was observed that Hb stays neither in its native state nor in its fully denatured state; rather, it achieves an intermediate state in the presence of ChAAILs. The research on the intermediate state of Hb is still unexplored. Research has been pursued to find a suitable ligand or IL that can stabilize the intermediate state of Hb. In that context, ChAAILs are among the best choices. Molecular docking studies unravel the binding of ChAAILs with Hb. The obtained binding energies of the docked complex are -7.2 kcal/mol and -8.7 kcal/mol for binding of Hb with [Chl][Gly] and [Chl][Met], respectively, which was in line with the ITC results. The quantum chemical calculations show that H-bond plays a significant role for the interaction between Hb and ChAAILs.

Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.

Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometre-scale resolution. With high-resolution images of predation in a native (hydrated, unstained) state, we discover several surprising features of the process, including macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following growth and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid.

Single-cell transcriptomics reveals maturation of transplanted stem cell–derived retinal pigment epithelial cells toward native state

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched invitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all invitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.

AMOEBA Force Field Predicts Accurate Hydrogen Bond Counts of Nitriles in SNase by Revealing Water-Protein Interaction in Vibrational Absorption Frequencies.

Precisely quantifying the magnitude and direction of electric fields in proteins has long been an outstanding challenge in understanding biological functions. Nitrile vibrational Stark effect probes have been shown to be minimally disruptive to the protein structure and can be better direct reporters of local electrostatic field in the native state of a protein than other measures such as pKa shifts of titratable residues. However, interpretations of the connection between measured vibrational energy and electric field rely on the accurate molecular understanding of interactions of the nitrile group and its environment, particularly from hydrogen bonding. In this work, we compared the extent of hydrogen bonding calculated in two common force fields, the fixed charge force field Amber03 and polarizable force field AMOEBA, at 10 locations of cyanocysteine (CNC) in staphylococcal nuclease (SNase) against the experimental nitrile absorption frequency in terms of full width at half-maximum (FWHM) and frequency temperature line slope (FTLS). We observed that the number of hydrogen bonds correlated well in AMOEBA trajectories with respect to both the FWHM (r = 0.88) and the FTLS (r = -0.85), whereas the correlation of Amber03 trajectories was less reliable because the Amber03 force field predicted more hydrogen bonds in some mutants. Moreover, we demonstrated that contributions from the interactions between CNC and nearby water molecules were significant in AMOEBA trajectories but were not predicted by Amber03. We conclude that although the nitrile absorption peak shape could be qualitatively predicted by the fixed charge Amber03 force field, the detailed electrostatic environment measured by the nitrile probe in terms of the extent of hydrogen bonding could only be accurately observed in the AMOEBA trajectories, where the permanent dipole, quadrupole, and dipole-induced-dipole polarizable interactions were all taken into account. The significance of this finding to the goal of accurately predicting electric fields in complex biomolecular environments is discussed.

Guided analysis of ambient ionization mass spectrometry data with the MQ_Assistant.

Ambient ionization mass spectrometry (AIMS) delivers realistic data from samples in their native state. In addition, AIMS methods reduce time and costs for sample preparation and have less environmental impact. However, AIMS data are often complex and require substantial processing before interpretation. We developed an interactive R script for guided mass spectrometry (MS) data processing. The "MQ_Assistant" is based on MALDIquant, a popular R package for MS data processing. In each step, the user can try and preview the effect of chosen parameters before deciding on the values with the best result and proceeding to the next stage. The outcome of the MQ_Assistant is a feature matrix that can be further analyzed in R and statistics tools such as MetaboAnalyst. Using 360 AIMS example spectra, we demonstrate the step-by-step processing for creating a feature matrix. In addition, we show how to visualize the results of three biological replicates of a plant-microbe interaction between Arabidopsis and Trichoderma as a heatmap using R and upload them to MetaboAnalyst. The final parameter set can be saved for reuse in MALDIquant workflows of similar data. The MQ_Assistant helps novices and experienced users to develop workflows for (AI)MS data processing. The interactive procedure supports the quick finding of appropriate settings. These parameters can be exported and reused in future projects. The stepwise operation with visual feedback also suggests the use of the MQ_Assistant in education.

Resolving the geometric structure of trastuzumab by mobility capillary electrophoresis and native mass spectrometry

Available online Immunoglobulins G (IgGs) are Y-shaped globular proteins, however, their high flexibility and heterogeneity pose great challenges to their structure and conformation determinations. Geometric structure of IgG closely correlates to its biofunctions, such as the antibody escape of HIV (human immunodeficiency virus) could attribute to the distance mismatch between the ends of two Fab arms (antigen-binding sites) and envelope glycoprotein spikes on virion surface. Herein, we report the first use of mobility capillary electrophoresis (MCE) and native mass spectrometry (nMS) to resolve the internal geometric structure and conformation of an IgG (trastuzumab) in solution phase. After proteolysis, the ellipsoid dimensions of IgG and its subunits were measured by MCE-nMS experiments. IgG was then reconstructed, in which the sizes and relative positions of these three subunits in three-dimensional space were characterized. It was found that the two Fab arms have an angle of ∼102.1° and a distance of ∼11.0 nm between the two antigen-binding sites under native condition, and the Fc arm was tilted ∼ 16.0° towards one of the Fab arms. Fc was not on the plane of Fab-Fab, but has an angle of no larger than 103.1°. Under acidic environment (pH 3.0), each subunit of the IgG would unfold into larger dimensions, and the angles between these subunits also change. With great potential for tumor imaging and therapy, the structure of F(ab′)2 fragments was also measured and validated by molecular dynamic simulation. It was found that the electrostatic force among these three subunits and steric hindrance stemming from Fc help maintaining the angle between two Fab arms.

Repurposing phytochemicals of Citrullus colocynthis against maltase-glucoamylase using molecular docking, MMGBSA, MD simulation and linear regression to identify potential anti-diabetic compounds

Diabetes is a common lifestyle disorder found in populations of different age groups. Maltase-glucoamylase catalyses the release of the glucose molecule in the final enzymatic reaction of starch digestion; therefore, inhibition of maltase-glucoamylase is one of the approaches in the development of therapeutics for diabetes. Citrullus colocynthis is commonly recommended in Ayurveda for the treatment of diabetes. The current study applied a structure-based drug design approach to repurpose the phytochemicals of Citrullus colocynthis to identify potential inhibitors for maltase-glucoamylase. 70 phytochemicals of Citrullus colocynthis were screened against maltase-glucoamylase and top 5 molecules 8-p-hydroxybenzylisovitexin, isoorientin, cucurbitacin B, cucurbitacin E, and cucurbitacin I with significant binding energy of −10 kcal/mol, −9.9 kcal/mol, −9.6 kcal/mol, −9.2 kcal/mol, and −7.7 kcal/mol were identified. Furthermore, MMGBSA, pharmacokinetics properties and toxicity prediction were performed on the five identified molecules and top 3 molecules were selected for molecular dynamics (MD) simulation. It was observed from the structural flexibility and dynamic behaviour of the systems that conformational changes were noticed in the complexes as compared to its native state, which suggests that the 3 molecules, namely 8-p-hydroxybenzylisovitexin, isoorientin, and cucurbitacin I of Citrullus colocynthis may act as inhibitors for maltase-glucoamylase. Communicated by Ramaswamy H. Sarma

Physiological Tensioning During Lower Trapezius Transfer for Irreparable Posterosuperior Rotator Cuff Tears May Be Important for Improvement in Shoulder Kinematics

Background: Lower trapezius transfer (LTT) has been proposed for restoring the anteroposterior muscular force couple in the setting of an irreparable posterosuperior rotator cuff tear (PSRCT). Adequate graft tensioning during surgery may be a factor critical for sufficient restoration of shoulder kinematics and functional improvement. Purpose/Hypothesis: The purpose was to evaluate the effect of tensioning during LTT on glenohumeral kinematics using a dynamic shoulder model. It was hypothesized that LTT, while maintaining physiological tension on the lower trapezius muscle, would improve glenohumeral kinematics more effectively than undertensioned or overtensioned LTT. Study Design: Controlled laboratory study. Methods: A total of 10 fresh-frozen cadaveric shoulders were tested using a validated shoulder simulator. Glenohumeral abduction angle, superior migration of the humeral head, and cumulative deltoid force were compared across 5 conditions: (1) native, (2) irreparable PSRCT, (3) LTT with a 12-N load (undertensioned), (4) LTT with a 24-N load (physiologically tensioned according to the cross-sectional area ratio of the lower trapezius muscle), and (5) LTT with a 36-N load (overtensioned). Glenohumeral abduction angle and superior migration of the humeral head were measured using 3-dimensional motion tracking. Cumulative deltoid force was recorded in real time throughout dynamic abduction motion by load cells connected to actuators. Results: Physiologically tensioned (Δ13.1°), undertensioned (Δ7.3°), and overtensioned (Δ9.9°) LTT each significantly increased the glenohumeral abduction angle compared with the irreparable PSRCT (P < .001 for all). Physiologically tensioned LTT achieved a significantly greater glenohumeral abduction angle than undertensioned LTT (Δ5.9°; P < .001) or overtensioned LTT (Δ3.2°; P = .038). Superior migration of the humeral head was significantly decreased with LTT compared with the PSRCT, regardless of tensioning. Physiologically tensioned LTT resulted in significantly less superior migration of the humeral head compared with undertensioned LTT (Δ5.3 mm; P = .004). A significant decrease in cumulative deltoid force was only observed with physiologically tensioned LTT compared with the PSRCT (Δ–19.2 N; P = .044). However, compared with the native state, LTT did not completely restore glenohumeral kinematics, regardless of tensioning. Conclusion: LTT was most effective in improving glenohumeral kinematics after an irreparable PSRCT when maintaining physiological tension on the lower trapezius muscle at time zero. However, LTT did not completely restore native glenohumeral kinematics, regardless of tensioning. Clinical Relevance: Tensioning during LTT for an irreparable PSRCT may be important to sufficiently improve glenohumeral kinematics and may be an intraoperatively modifiable key variable to ensure postoperative functional success.

Polyampholytes and Their Hydrophobic Derivatives as Excipients for Suppressing Protein Aggregation.

Protein aggregation, which occurs under various physiological conditions, can affect cell function and is a major issue in the field of protein therapeutics. In this study, we developed a polyampholyte composed of ε-poly-l-lysine and succinic anhydride and evaluated its protein protection efficacy. This polymer was able to protect different proteins from thermal stress and its performance significantly exceeded that of previously reported zwitterionic polymers. In addition, we synthesized derivatives with varying degrees of hydrophobicity, which exhibited remarkably enhanced efficiency; thus, the polymer concentration required for protein protection was very low. By facilitating the retention of protein enzymatic activity and stabilizing the higher-order structure, these polymers enabled the protein to maintain its native state, even after being subjected to extreme thermal stress. Thus, such polyampholytes are extremely effective in protecting proteins from extreme stress and may find applications in protein biopharmaceuticals and drug delivery systems.

Front Cover: Chemical and Biological Strategies for Profiling Protein‐Protein Interactions in Living Cells (Chem. Asian J. 14/2023)

Elucidation of Protein-Protein Interactions (PPIs) in living cells/animals is very important for the study of basic biology and the development of novel therapeutics. Recent advances in the techniques for profiling PPIs, such as fluorescence microscopy and various proximity labeling-based methods coupled with mass spectrometry (MS), have provided such a great opportunity to study PPIs in their native states with spatiotemporal resolution, especially the ones that are inaccessible to conventional co-immunoprecipitation (co-IP) and pull-down assays. More information can be found in the Review by You-Yu Wang, Wenyi Li, Bang-Ce Ye, and Xiao-Bao Bi.

Effects of Hydrogen/Deuterium Exchange on Protein Stability in Solution and in the Gas Phase.

Mass spectrometry (MS)-based techniques are widely used for probing protein structure and dynamics in solution. H/D exchange (HDX)-MS is one of the most common approaches in this context. HDX is often considered to be a "benign" labeling method, in that it does not perturb protein behavior in solution. However, several studies have reported that D2O pushes unfolding equilibria toward the native state. The origin, and even the existence of this protein stabilization remain controversial. Here we conducted thermal unfolding assays in solution to confirm that deuterated proteins in D2O are more stable, with 2-4 K higher melting temperatures than unlabeled proteins in H2O. Previous studies tentatively attributed this phenomenon to strengthened H-bonds after deuteration, an effect that may arise from the lower zero-point vibrational energy of the deuterated species. Specifically, it was proposed that strengthened water-water bonds (W···W) in D2O lower the solubility of nonpolar side chains. The current work takes a broader view by noting that protein stability in solution also depends on water-protein (W···P) and protein-protein (P···P) H-bonds. To help unravel these contributions, we performed collision-induced unfolding (CIU) experiments on gaseous proteins generated by native electrospray ionization. CIU profiles of deuterated and unlabeled proteins were indistinguishable, implying that P···P contacts are insensitive to deuteration. Thus, protein stabilization in D2O is attributable to solvent effects, rather than alterations of intraprotein H-bonds. Strengthening of W···W contacts represents one possible explanation, but the stabilizing effect of D2O can also originate from weakened W···P bonds. Future work will be required to elucidate which of these two scenarios is correct, or if both contribute to protein stabilization in D2O. In any case, the often-repeated adage that "D-bonds are more stable than H-bonds" does not apply to intramolecular contacts in native proteins.

An Activatable Nanoscintillator Probe for Detecting Telomerase Activity and Screening Inhibitors In Vivo

Telomerase represents an essential molecular machinery for tumor occurrence and progression and a potential therapeutic target for cancer treatment. Sensitive and reliable analysis of telomerase activity is of significant importance for the diagnosis and treatment of cancer. In this study, we developed a telomerase-activated nanoscintillator probe for deep-tissue and background-free imaging of telomerase activity and screening telomerase inhibitors in tumor-bearing living mice models. The probe was constructed by modifying lanthanide-doped nanoscintillators with aptamer-containing DNA anchor strands which hybridized with quencher labelled–oligonucleotide strands and telomerase primers. The X-ray-induced fluorescence of the probe was quenched originally but turned on upon telomerase-catalyzed extension of the primer. Benefiting from exceptional tissue penetrating properties and negligible autofluorescence of X-ray excitation, this probe enabled direct detection of telomerase activity in vivo via fluorescence imaging. Furthermore, with the direct, readable fluorescent signals, the probe enabled the screening of telomerase inhibitors in living cells and whole-animal models in the native states of telomerase. This strategy would inspire the development of low autofluorescence and deep tissue bioimaging probes for disease diagnosis and drug development in high-level living settings.

Capillary electrophoresis-mass spectrometry for protein analyses under native conditions: Current progress and perspectives.

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigration separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out.

A forgotten famine of ’43? Travancore’s muffled ‘cry of distress’

AbstractMass disease and starvation in the princely state of Travancore during the Second World War claimed some 90,000 lives. However, this episode has never received much prominence, especially when compared to the simultaneous crisis in Bengal. It is, in many ways, forgotten. Instead, Travancore’s wartime food management apparatus appears in some accounts as a success story. How did this happen? Integration into the world economy, the reordering of a rigid social structure, and popular political pressures on an autocratic princely regime created a unique set of conditions that left Travancore vulnerable to food scarcity and conflict during the Second World War. A particularly draconian princely regime that suppressed civil liberties prevented the gravity of the situation from being understood. This culminated in vastly unequal suffering and disease-related deaths. But the story is not merely one of despair. The Indian communists took advantage of war conditions to bring together agricultural and factory labourers and contribute to improving the food situation in this ‘People’s War’, while mainstream nationalists sought to obstruct the war effort and have the British ‘quit’ India. Wartime activities would shape the unique post-colonial politics of what became the state of Kerala in 1956. Intervening at the intersection of the historiographies of food and the princely states, this article adds a regional perspective to the nation-centric social history of the Second World War in South Asia. Hunger was a constitutive experience of this period across various parts of India, but the post-colonial political legacies of war could be regionally distinct.

Structural analysis of the water channel AQP2 by single-particle cryo-EM

Water channels, which are small membrane proteins almost entirely buried in lipid membranes, are challenging research targets for single-particle cryo-electron microscopy (cryo-EM), a powerful technique routinely used to determine the structures of membrane proteins. Because the single-particle method enables structural analysis of a whole protein with flexible parts that interfere with crystallization, we have focused our efforts on analyzing water channel structures. Here, utilizing this system, we analyzed the structure of full-length aquaporin-2 (AQP2), a primary regulator of vasopressin-dependent reabsorption of water at the renal collecting ducts. The 2.9 Å resolution map revealed a cytoplasmic extension of the cryo-EM density that was presumed to be the highly flexible C-terminus at which the localization of AQP2 is regulated in the renal collecting duct cells. We also observed a continuous density along the common water pathway inside the channel pore and lipid-like molecules at the membrane interface. Observations of these constructions in the AQP2 structure analyzed without any fiducial markers (e.g., a rigidly bound antibody) indicate that single-particle cryo-EM will be useful for investigating water channels in native states as well as in complexes with chemical compounds.

Quantum-based modeling implies that bidentate Arg89-substrate binding enhances serine/threonine protein phosphatase-2A(PPP2R5D/PPP2R1A/PPP2CA)-mediated dephosphorylation.

PP2A-serine/threonine protein phosphatases function as heterotrimeric holoenzymes, composed of a common scaffold (A-subunit encoded by PPP2R1A/PPP2R1B), a common catalytic (C-subunit encoded by PPP2CA/PPP2CB), and one of many variable regulatory (B) subunits. The site of phosphoprotein phosphatase (PPP) hydrolysis features a bimetal system (M1/M2), an associated bridge hydroxide [W1(OH-)], and a highly-conserved core sequence. In the presumptive common mechanism, the phosphoprotein's seryl/threonyl phosphate coordinates the M1/M2 system, W1(OH-) attacks the central P atom, rupturing the antipodal bond, and simultaneously, a histidine/aspartate tandem protonates the exiting seryl/threonyl alkoxide. Based on studies of PPP5C, a conserved arginine proximal to M1 is also expected to bind the substrate's phosphate group in a bidentate fashion. However, in PP2A isozymes, the role of the arginine (Arg89) in hydrolysis is not clear because two independent structures for PP2A(PPP2R5C) and PP2A(PPP2R5D) show that Arg89 engages in a weak salt bridge at the B:C interface. These observations raise the question of whether hydrolysis proceeds with or without direct involvement of Arg89. The interaction of Arg89 with B:Glu198 in PP2A(PPP2R5D) is significant because the pathogenic E198K variant of B56δ is associated with irregular protein phosphorylation levels and consequent developmental disorders (Jordan's Syndrome; OMIM #616355). In this study, we perform quantum-based hybrid [ONIOM(UB3LYP/6-31G(d):UPM7)] calculations on 39-residue models of the PP2A(PPP2R5D)/pSer (phosphoserine) system to estimate activation barriers for hydrolysis in the presence of bidentate Arg89-substrate binding and when Arg89 is otherwise engaged in the salt-bridge interaction. Our solvation-corrected results yield ΔH‡ ≈ ΔE‡ = +15.5kcal/mol for the former case, versus +18.8kcal/mol for the latter, indicating that bidentate Arg89-substrate binding is critical for optimal catalytic function of the enzyme. We speculate that PP2A(PPP2R5D) activity is suppressed by B:Glu198 sequestration of C:Arg89 under native conditions, whereas the PP2A(PPP2R5D)-holoenzyme containing the E198K variant has a positively-charged lysine in this position that alters normal function.