Parapharyngeal Nodal Metastasis in Papillary Thyroid Carcinoma: Catching Early.

Background: Metastasis to parapharyngeal space in papillary carcinoma of the thyroid is rare and often missed in routine clinical and radiological investigations. Although contrast CT /MRI can be done to locate the parapharyngeal lesion, the diagnosis of metastasis mostly deepened upon aspiration cytology, which is a challenge in difficult anatomical locations. Here, we have emphasized the management dilemma of parapharyngeal metastasis in papillary carcinoma of the thyroid. Case Report: A 30-year-old female presented with multiple right-side neck swelling for two months. Ultrasonography of right thyroid showed TIRAD V and left thyroid showed TIRAD II. Aspiration cytology of the right thyroid showed Bethesda III and aspiration cytology revealed thyroid follicles. Due to the diagnostic ambiguity, contrast-enhanced CT scan was advised. It revealed a hypervascular lymph node in the parapharyngeal space besides multiple right cervical lymph nodes and later changed the treatment plan. Conclusion: - Metastasis to parapharyngeal space in Papillary carcinoma of the thyroid is rare and often missed on routine evaluation. Contrast-enhanced CT/MRI can be added as a primary investigation in challenging cases of suspicious malignancy of the thyroid.

To evaluate the efficacy of topical anti-fungal therapy in postoperative cases of COVID Associated Mucormycosis (CAM): A single-blinded randomized control trial.

PURPOSE: To compare the efficacy of 0.1 % w/w Liposomal Amphotericin-B gel with 10 % w/w Povidone-Iodine and saline nasal douching in preventing revision surgery in patients with CAM. STUDY DESIGN: Multi-arm, parallel randomized control trial. STUDY SETTING: The trial was conducted in the Department of ENT, All India Institute of Medical Sciences (AIIMS) Bhubaneswar. METHODS: Participants: Microbiologically and histologically proven cases of mucormycosis who underwent surgical debridement were included in the study. INTERVENTIONS: Postoperatively, patients were randomized into three groups based on the type of topical intervention received, in the form of Lipid-based Amphotericin B gel, povidone­iodine ointment or saline nasal douching. OUTCOME: Requirement of revision surgery in postoperative cases of CAM. RANDOMIZATION: Participants were allotted to one of the three arms by block randomization. BLINDING: Single-blinded trial. RESULTS: Numbers randomized: 15 participants were randomized to each group. Recruitment: Completed recruiting. Numbers analyzed: 15 participants were analyzed in each group. OUTCOMES: Control arm's risk of revision surgery was 4.50 (95 % CI: 1.16-17.44) times than Lipid-based Amphotericin B gel arm and 1.50 (95 % CI: 0.71-3.16) times that of the Povidone- Iodine arm. The difference was statistically significant (p = 0.02) for Amphotericin but not for Povidone-Iodine. CONCLUSIONS: Topical Amphotericin-B gel application in the postoperative cavity can decrease the need for revision surgery and help in early recovery. TRIAL REGISTRATION: CTRI/2021/10/037257. Clinical Trials Registry of India.

Predisposing factors of rhino-orbital-cerebral mucormycosis in patients with COVID 19 infection.

The predisposing factors of invasive fungal disease in COVID 19 infection are still debatable because of the limited human understanding of the virus with the current literature. In this study, we have tried to correlate the various predisposing factors influencing the clinical profile and treatment outcomes in patients with covid associated mucormycosis (CAM). It is a retrospective analysis of cases of CAM during the second wave of COVID 19 infection, which was managed in the department of Otorhinolaryngology from Dec 1, 2020, to June 10, 2021. The detailed clinical, radiological and management of patients with CAM were collected, recorded, evaluated and correlated with the predisposing factors. Of the total, 46 patients, 44(95.65%) were diabetic and 41 patients had a previous history of steroid intake. When clinical parameters were compared between blood sugar < 200 mg/dl and > 200 mg/dl, the old and newly diagnosed diabetes mellitus in patients with CAM, there was no significant differences in any of the above clinical parameters (p > 0.05), except the hospital stay (p = 0,004). Steroid intake in patients with coexisting DM associated with CAM is considered the most important factor for the development of the CAM. There was are no significant difference in any of the clinical/treatment outcomes in patients with CAM with respect to the initial blood sugar, except for the hospital stay. A large sample size with a long-term follow-up period may be needed for a better understanding of common predisposing factors for the development of CAM.

Vulnerability of groundwater from elevated nitrate pollution across India: Insights from spatio-temporal patterns using large-scale monitoring data.

Agriculture-sourced, non-point groundwater contamination (e.g., nitrate) is a serious concern from the drinking water crisis aspect across the agrarian world. India is one of the largest consumers of nitrogen fertilizers in South-Asia as well as in the world but groundwater nitrate lacks critical attention as a wide-scale drinking water pollutant in the country. Our study provides the first documentation of the distribution of groundwater nitrate and the extent of elevated nitrate contamination across India, along with the delineation of the temporal trends and the natural and anthropogenic factors that influence such occurrence of groundwater nitrate. High resolution, annual-scale spatio-temporal variability of groundwater nitrate concentration and consequent contamination was delineated using groundwater nitrate measurements from ~3 million drinking water wells spread across 7038 administrative blocks between 2010 and 2017 in India. An average 8% of the studied blocks were found affected by elevated groundwater nitrate (> 45 mg/L). Depth-dependent trend demonstrated that nitrate concentrations were about 14% higher in shallow water wells (≤ 35 m) than deep wells (>35 m). The overall temporal trend of groundwater nitrate concentration was decreasing slightly nationwide in the study period. The correlation tests and causality test results indicated that the spatial distribution of groundwater nitrate was significantly associated with agricultural N-fertilizer usage, whereas the decreasing temporal trend corresponded with the overall reduced N-fertilizer usage during the study period. Spatial autocorrelation analysis identified the clustering of high nitrate areas in central, north, and southern India, specifically in areas with higher fertilizer usage. We estimate about 71 million Indians possibly exposed to elevated groundwater nitrate concentrations and the majority of them reside in rural areas. Thus, this study provides the previously unrecognized, wide-scale, anthropogenic, diffused groundwater nitrate contamination across India.

Achieving Sustainable Development Goal for Clean Water in India: Influence of Natural and Anthropogenic Factors on Groundwater Microbial Pollution.

Worldwide, >2 billion people (~1/3 world population), mostly living in economically stressed areas of Africa and South Asia, still do not have access to basic sanitation, and ~1 billion still practice open defecation. Water pollution due to open defecation may primarily be linked to economy, and other factors such as social and hygiene practices, land use and hydrogeological parameters could also have sufficient influence. The present study describes the effect of human development index (HDI, 2001-2015) and economic development (NL, 1992-2013) on groundwater microbial pollution (FC, 2002-2017) across India. Economic development pattern suggested discernable inverse relationship with FC in most areas, although areas with inferior water quality, improper human practices were found to outweigh economic development. Vulnerability modelling, using these data, along with measured FC in groundwater-sourced drinking water locations (n = 235) demonstrated the heterogeneity of FC distribution potential in areas of homogenous economy, social practices, and land use. High-resolution numerical modelling of the advective transport of the hypothetical FC particles in the aquifers, suggest up to ~24 times faster movement of pollutants under irrigation-induced pumping regimes. Hence, the results of our study highlight and quantify the potential pitfalls that are possible hindrance for achieving the United Nations sustainable development goal, despite social and economic development, across the spatial scales.

Author Correction: Impact of sanitation and socio-economy on groundwater fecal pollution and human health towards achieving sustainable development goals across India from ground-observations and satellite-derived nightlight.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Impact of sanitation and socio-economy on groundwater fecal pollution and human health towards achieving sustainable development goals across India from ground-observations and satellite-derived nightlight.

Globally, ~1 billion people, mostly residing in Africa and South Asia (e.g. India), still lack access to clean drinking water and sanitation. Resulting, unsafe disposal of fecal waste from open-defecation to nearby drinking water sources severely endanger public health. Until recently, India had a huge open-defecating population, leading declining public health from water-borne diseases like diarrhoea by ingesting polluted water, mostly sourced to groundwater. However, in recent past, sanitation development to achieve Sustainable Development Goals (SDGs) has been encouraged throughout India, but their effect to groundwater quality and human health conditions are yet-unquantified. Here, for the first time, using long term, high-spatial resolution measurements (>1.7 million) across India and analyses, we quantified that over the years, groundwater fecal coliform concentration (2002-2017, -2.56 ± 0.06%/year) and acute diarrheal cases (1990-2016, -3.05 ± 0.01%/year) have significantly reduced, potentially influenced by sanitation development (1990-2017, 2.63 ± 0.01%/year). Enhanced alleviation of groundwater quality and human health have been observed since 2014, with initiation of acceletated constructions of sanitation infrastructures through Clean India (Swachh Bharat) Mission. However, the goal of completely faecal-pollution free, clean drinking water is yet to be achieved. We also evaluated the suitability of using satellite-derived night-time light (NLan, 1992-2013, 4.26 ± 0.05%/year) as potential predictor for such economic development. We observed that in more than 80% of the study region, night-time light demonstrated to be a strong predictor for observed changes in groundwater quality, sanitation development and water-borne disease cases. While sanitation and economic development can improve public health, poor education level and improper human practices can strongly influence on water-borne diseases loads and thus health in parts of India.

Selective lighting up of segments around Gly, Ala and Ser/Thr in proteins.

Direct detection of (13) C nucleus can be used as a valuable alternative where (1) H detection poses a challenge due to relaxation effects, chemical exchange and poor chemical shift dispersion. In this context, we have designed a suite of 2D (13) C(α) -detected hNCA experiments that provide sequential correlations of (13) C(α) with (15) N on one hand and efficient spectroscopic labeling of certain groups of residues, namely, Gly, Ala, Ser and Thr, on the other. These residues act as checkpoints in the sequential walk, which in turn offer new possibilities of backbone assignment of small proteins from a set of 2D experiments, thereby providing great economy in terms of spectrometer time. The direct identification of peptide segments around Gly, Ala, Ser and Thr residues along a protein chain will be highly valuable for deriving important information on sites of ligand binding, phosphorylation, inhibitor/substrate binding, understanding protein folding pathways, comprehending local conformational dynamics etc. without having to obtain complete sequence-specific assignments, which can be time consuming and at times formidable, especially in large proteins. We have illustratively demonstrated the multifaceted applications of these variants of 2D experiments on ubiquitin and M-crystallin. We foresee that these 2D hNCA experiments will provide economic and efficient strategies for studying the structure and function of proteins.

Intrinsic vs environment driven equilibrium folding transitions in GTPase effector domain of dynamin: NMR insights.

Relative importance of the intrinsic properties of the polypeptide chain vis-a-vis the environmental influences, in driving the folding of a protein, has been a subject of extensive debate and investigation. Folding/misfolding lead to self association in many systems, which have biological functional significance. We compare here, the NMR derived equilibrium folding transitions driven under different environmental conditions in the GTPase Effector Domain of dynamin, which self-associates into megadalton size species. We conclude that though hierarchy of folding and association of GED is substantially influenced by the solvents, these properties, to a good extent are also driven by intrinsic properties of the polypeptide chain, and the regions that form secondary structures, the types of secondary structures formed in those regions, and finally the regions that participate in the self-association are the same, indicating near neighbor interactions would have a telling effect on the final outcome of the folding process. These observations would open a new reliable frontier for elucidating the multiple folding trajectories and consequent self-association, by simulations in vacuum, for this protein.

NMR derived model of GTPase effector domain (GED) self association: relevance to dynamin assembly.

Self-association of dynamin to form spiral structures around lipidic vesicles during endocytosis is largely mediated by its 'coiled coil' GTPase Effector Domain (GED), which, in vitro, self-associates into huge helical assemblies. Residue-level structural characterizations of these assemblies and understanding the process of association have remained a challenge. It is also impossible to get folded monomers in the solution phase. In this context, we have developed here a strategy to probe the self-association of GED by first dissociating the assembly using Dimethyl Sulfoxide (DMSO) and then systematically monitoring the refolding into helix and concomitant re-association using NMR spectroscopy, as DMSO concentration is progressively reduced. The short segment, Arg109 - Met116, acts as the nucleation site for helix formation and self-association. Hydrophobic and complementary charge interactions on the surfaces drive self-association, as the helices elongate in both the directions resulting in an antiparallel stack. A small N-terminal segment remains floppy in the assembly. Following these and other published results on inter-domain interactions, we have proposed a plausible mode of dynamin self assembly.

Residual structure and dynamics in DMSO-d6 denatured dynein light chain protein.

Structural and motional features in the denatured state of a protein dictate the early folding events starting from that state and these features vary depending upon the nature of the denaturant used. Here, we have attempted to decipher the early events in the folding of Dynein Light Chain protein (DLC8), starting from DMSO-d6 denatured state. Multinuclear NMR experiments were used to obtain the full spectral assignment. The HSQC spectrum shows the presence of two sets of peaks for the residues Met 1, Ser 2, Arg 4, Ala 11, Met 17, Thr 26, Lys 44, Tyr 50, Asn 51, Trp 54, His 55, Val 58, Gly 59, Ser 64, Tyr 65, His 68, Phe 86, Lys 87 indicating the presence of slow conformational transition in the heterogeneous ensemble. Analysis of residual structural propensities with secondary (13)C chemical shifts, (3)J(H(N)(-)H(α)) coupling constants and (1)H-(1)H NOE revealed the presence of local preferences which encompass both native and non-native like structures. The spectral density calculations, as obtained from measured R(1), R(2) and (1)H-(15)N steady state NOE values provide insights into the backbone dynamics on the milli to picosecond timescale. The segment Ser 14 - His 55 exhibits slow motions on the milli- to microsecond timescale arising from conformational exchange. The presence of native like structural preference, as well as conformational exchange classifies the above segment as the nucleation site of folding. Based on the observations, we propose here, the probable hierarchy of folding of DLC8 on dilution of denaturant: the two helices are formed first followed by the formation of ß2 and ß5.

Simultaneous acquisition of 13Cα-15N and 1H-15N-15N sequential correlations in proteins: application of dual receivers in 3D HNN.

We describe here, adaptation of the HNN pulse sequence for multiple nuclei detection using two independent receivers by utilizing the detectable (13)C(α) transverse magnetization which was otherwise dephased out in the conventional HNN experiment. It enables acquisition of 2D (13)C(α)-(15)N sequential correlations along with the standard 3D (15)N-(15)N-(1)H correlations, which provides directionality to sequential walk in HNN, on one hand, and enhances the speed of backbone assignment, on the other. We foresee that the implementation of dual direct detection opens up new avenues for a wide variety of modifications that would further enhance the value and applications of the experiment, and enable derivation of hitherto impossible information.

The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures.

As yeast cultures enter stationary phase in rich, glucose-based medium, differentiation of two major subpopulations of cells, termed quiescent and nonquiescent, is observed. Differences in mRNA abundance between exponentially growing and stationary-phase cultures and quiescent and nonquiescent cells are known, but little was known about protein abundance in these cells. To measure protein abundance in exponential and stationary-phase cultures, the yeast GFP-fusion library (4159 strains) was examined during exponential and stationary phases, using high-throughput flow cytometry (HyperCyt). Approximately 5% of proteins in the library showed twofold or greater changes in median fluorescence intensity (abundance) between the two conditions. We examined 38 strains exhibiting two distinct fluorescence-intensity peaks in stationary phase and determined that the two fluorescence peaks distinguished quiescent and nonquiescent cells, the two major subpopulations of cells in stationary-phase cultures. GFP-fusion proteins in this group were more abundant in quiescent cells, and half were involved in mitochondrial function, consistent with the sixfold increase in respiration observed in quiescent cells and the relative absence of Cit1p:GFP in nonquiescent cells. Finally, examination of quiescent cell-specific GFP-fusion proteins revealed symmetry in protein accumulation in dividing quiescent and nonquiescent cells after glucose exhaustion, leading to a new model for the differentiation of these cells.

Resonance assignments of GTPase effector domain of dynamin in the aprotic solvent deuterated dimethyl sulfoxide.

The GTPase effector domain (GED) is a subunit of dynamin, a multi-domain protein involved in endocytosis. GED forms a megadalton-sized self-assembly in vitro. The core of such huge assemblies is inaccessible to detailed Nuclear Magnetic Resonance characterization by conventional methods due to line broadening effects. Till date, there have been no studies to directly identify the residues involved in the core of the assembly. In this background we report here the NMR resonance assignments of deuterated dimethyl sulfoxide (DMSO-d6)-denatured GED from Homo sapiens. This will form the basis for probing the core of GED assembly and characterization of the association pathway driven by DMSO dilution.

NMR insights into the core of GED assembly by H/D exchange coupled with DMSO dissociation and analysis of the denatured state.

GTPase effector domain (GED) of dynamin forms megadalton-sized assembly in vitro, rendering its structural characterization highly challenging. To probe the core of the GED assembly, we performed H/D exchange in native state and analyzed the residual amides following dissociation by dimethyl sulfoxide (DMSO). The data indicated a hierarchy in solvent exposure: Ser2-Glu13, Glu23-Phe32, Asp37-Gln43, Val51-Met55, and Lys60-Asp64 followed by the remaining segments. This reflects the chain packing in the core of the assembly. The segment Leu65-Pro138 in the C-terminal half is largely in the interior of the core, while the N-terminal segment Asp37-Asp64 traverses into and out of the core. Next, we characterized the structural and motional behavior of the DMSO-denatured state. The stretches Gly9-Lys18, Asp37-Arg42, Lys68-Met74, and Ser136-Thr137 were seen to display alternate conformations in slow exchange. In the major population, both α and ß propensities were seen along the polypeptide chain. Spectral density analysis of (15)N R(1), R(2), and (1)H-(15)N nuclear Overhauser effect collected at 600 and 800 MHz suggested the presence of four domains of slow motions, namely, A (Leu40-Tyr91), A' (Leu124-Ile130); B (Asn97-Gln107), B' (Tyr117-Leu120), two of which flank the region Arg109-Met116, for which no peaks are seen in the heteronuclear single quantum coherence spectrum. These domains would identify folding and association initiation sites of GED. Interestingly, they also coincide with the helical domain in the native state, suggesting that helix formation leads to self-association of GED.

Hierarchy of local structural and dynamics perturbations due to subdenaturing urea in the native state ensemble of DLC8 dimer.

Local structural and dynamic modulations due to small environmental perturbations reflect the adaptability of the protein to different interactors. We have investigated here the preferential local perturbations in Dynein light chain protein (DLC8), a cargo adapter, by sub-denaturing urea concentrations. Equilibrium unfolding experiments by optical spectroscopic methods indicated a two state like unfolding of DLC8 dimer, with the transition mid-point occurring around 8.6M urea. NMR studies identified the ß3 and ß4 strands, N-, C- terminal regions, loops connecting ß1 to α1, α1 to α2 and ß3 to ß4 as the soft targets of urea perturbation and thus indicated potential unfolding initiation sites. Native-state hydrogen exchange studies suggested the unfolding to traverse from the edges towards the centre of the secondary structural elements. At 6M urea the whole protein chain acts like a cooperative unit. These observations are expected to have important implications for the protein's multiple functions.

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange.

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65 degrees C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein.

Residue-wise conformational stability of DLC8 dimer from native-state hydrogen exchange.

Dynein light chain (DLC8) is the smallest subunit of the dynein motor complex, which is known to act as a cargo adaptor in intracellular trafficking. The protein exists as a pure dimer at physiological pH and a completely folded monomer below pH 4. Here, we have determined the energy landscape of the dimeric protein using a combination of optical techniques and native-state hydrogen exchange of amide groups, the former giving the global features and the latter yielding the residue level details. The data indicated the presence of intermediates along the equilibrium unfolding transition. The hydrogen exchange data suggested that the molecule has differential stability in its various segments. We deduce from the free energy data that the antiparallel beta-sheets (beta4 and beta5) that form the hydrophobic core of the protein and the alpha2 helix, all of which are highly protected with regard to hydrogen exchange, contribute significantly to the initial step of the protein folding mechanism. Denaturant-dependent hydrogen exchange indicated further that some amides exchange via local fluctuations, whereas there are others which exchange via global unfolding events. Implications of these to cargo adaptability of the dimer are discussed.