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SummaryInoviruses are abundant filamentous phages infecting numerous prokaryotic phyla, where they can symbiotically promote host fitness and increase bacterial virulence. Due to their unique properties, inoviruses have also been utilised in biotechnology for phage display and as models for studying phase behaviour of colloidal rods. Inoviral phages secreted by bacteria can self-assemble into liquid crystalline droplets that protect bacterial cells in biofilms from antibiotics, however, factors governing the formation of such droplets and the mechanism of antibiotic protection are poorly understood. Here, we investigate the structural, biophysical, and protective properties of liquid crystalline droplets formed byPseudomonas aeruginosaandEscherichia coliinoviral phages. We report a cryo-EM structure of the capsid from the highly studiedE. colifd phage, revealing distinct biochemical properties of fd compared to Pf4 phage fromP. aeruginosa. We show that fd and Pf4 form liquid crystalline droplets with diverse morphologies governed by the underlying phage particle geometry and biophysics, rather than their surface biochemical properties. Finally, we show that these morphologically diverse droplets made of either phage can protect rod-shaped bacteria from antibiotic treatment, despite differing modes of association with cells. This study advances our understanding of phage assembly into liquid crystalline droplets, and provides insights into how filamentous molecules protect bacteria from extraneous molecules under crowding conditions, which are found in biofilms or on infected host tissues.

The study was aimed to assess the psychological aspects and relevant factors of the health-care workers (HCWs) working in COVID 19 pandemic condition in Bangladesh. This online cross-sectional survey was conducted from different tertiary, secondary and primary hospitals in Bangladesh. Eligible 638 HCWs who were directly involved in the caring of confirmed or suspected COVID-19 patients were recruited in this study. The mental health was assessed by the Patient Health Questionnare-9 (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7) and Athens Insomnia Scale (AIS). High frequency of depression 536(84.0%), anxiety 386(60.5%) and insomnia 302(47.3%) was found among the HCWs, which were significantly higher in physicians (p0.001) than nurses. Moderate to severe depression was significantly higher in female, whereas minimal to mild depression was significant in male HCWs (p=0.014). Symptoms of depression (p0.001), anxiety (p0.001) and insomnia (p=0.004) were significantly higher among the HCWs of primary and secondary compared to the tertiary level. The HCWs developed psychological trauma due to family health (45.3%) and contagious disease property (66.6%). After adjusting confounders, multivariable logistic regression analysis showed that physicians and HCWs of secondary hospital had significant symptoms of severe depression (OR=2.95, 95% CI=0.50-17.24; p0.001), anxiety (OR=2.64, 95% CI=0.80-8.72; p0.001) and insomnia (OR=2.67, 95% CI=1.23-5.84; p=0.018); whereas female HCWs had more risk of developing symptoms of severe insomnia (OR= 1.84; 95% CI=1.23-2.75; p=0.003). High rate of depression, anxiety and insomnia was found among HCWs working in the COVID-19 pandemic condition in this survey.

In this report, serious negative interference of Dissolved Organic Compounds (DOCs) in the laser/LED fluorimetric determination of uranium in natural and waste waters has been amply demonstrated. The experiments exploring the effects of the concentration of individual DOCs on the fluorescence intensity of a fixed concentration of uranium(VI) were designed involving about 15 different DOCs, mostly being the degradation products of humic acid and lignin, and the corresponding results obtained were compared with the results obtained by applying the method to a host of (about 500) real natural water samples sampled from areas of diverse geological and environmental settings. The results were compared with those obtained by other standard methods. In most cases, the fluorescence signal increases substantially on destruction of the DOCs present in the water samples studied, depending on the contents of the DOCs and their respective complexing abilities. Woefully, determination of low contents of uranium in some of the natural water samples by the hitherto recommended/prescribed LIF or LED fluorimetric methods often entails a great deal of difficulty in the interpretation of analytical results obtained, in spite of the metal ion (uranium) being actually present in them in significant quantities. Based on the above study, an improved LIF/LED fluorimetric method has been developed, which is robust and highly effective for accurate and precise determination of uranium(VI) in any type of water sample.

A novel and useful extraction spectrophotometric method for the rapid determination of uranium and free acidity in nuclear fuel process samples using the reagent, 2,3-dihydroxynaphthalene as an extractant as well as color forming agent is described. Uranium at milligram level forms a yellowish- orange anionic complex with the ligand, 2,3-dihydroxynaphthalene over a pH range, 10–12. This anionic complex is easily extracted into ethylacetate as an ion-pair complex with cetyltrimethylammonium cation (CTA+). The absorbance of the ion-pair complex in ethylacetate is measured at 390 nm. The molar absorptivity and Sandell’s sensitivity of the system being 5.0×103 L·mol−1 cm−1 and 0.047 μg cm−2, respectively at 390 nm. This very reagent (2,3-dihydroxynaphthalene) also forms a yellowish-orange nitro product with free nitric acid (HNO3) in the presence of concentrated sulfuric acid. This nitro- product is easily extracted into the same solvent (ethylacetate) and absorbs maximum at 380 nm. The molar absorptivity (ε) and Sandell’s sensitivity of the system being 2.4×103 L·mol−1 cm−1 and 0.027 μg cm−2, respectively. By using the single chromophoric agent (2,3-dihydroxynaphthalene), both uranium and free acidity can be easily and reliably determined separately in the process solution. The relative standard deviations (RSD) are in the range, 0.5–2.0% and 1.5–2.0%, respectively for uranium and free nitric acid determinations. The method has been applied to nuclear fuel process solutions, and the results obtained have been found to be favorably comparable with those obtained from standard methods.

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of antibiotic-tolerant infections in humans. P. aeruginosa evades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral prophage, Pf4. We investigated the mechanism of phage-mediated antibiotic tolerance using biochemical reconstitution combined with structural biology and high-resolution cellular imaging. We resolved electron cryomicroscopy atomic structures of Pf4 with and without its linear single-stranded DNA genome, and studied Pf4 assembly into liquid crystalline droplets using optical microscopy and electron cryotomography. By biochemically replicating conditions necessary for antibiotic protection, we found that phage liquid crystalline droplets form phase-separated occlusive compartments around rod-shaped bacteria leading to increased bacterial survival. Encapsulation by these compartments was observed even when inanimate colloidal rods were used to mimic rod-shaped bacteria, suggesting that shape and size complementarity profoundly influences the process. Filamentous inoviruses are pervasive across prokaryotes, and in particular, several Gram-negative bacterial pathogens including Neisseria meningitidis , Vibrio cholerae, and Salmonella enterica harbor these prophages. We propose that biophysical occlusion mediated by secreted filamentous molecules such as Pf4 may be a general strategy of bacterial survival in harsh environments.

An extractive pellet fluorimetry determination of trace uranium in thorium rich samples has been developed. This is based upon a solvent extraction system which completely separates both the elements uranium and thorium from each other. Thorium as a neutral complex with 2,3-dihydroxynaphthalene at pH 4–6 is extracted into ethylacetate and then uranium-2,3-dihydroxynaphthalene anionic complex is extracted into another batch of ethylacetate at pH 11–12 under the influence of a counter cation, cetyltrimethylammonium ion. This method has been applied for the determination of trace uranium in synthetic nuclear grade thorium oxide and thorium rich mineralized rock with high degree of accuracy and precision.

A method for precise and accurate determination of ppm to percentage level uranium in rocks, soils and stream sediments by laser induced fluorimetry (LIF) has been developed. Iron, above a ratio (iron to uranium) of 100, which is always the case when uranium is present at ppm level, interferes seriously. A prior removal of iron enables the determination of uranium over a wide range (ppm to % level).The method has been successfully applied to a host of silicate rock, soil and sediment samples, and the results obtained have been validated by conventional pellet fluorimetry after pre-concentration of the analyte.

Epigenetic information is transmitted from mother to daughter cells through mitosis. To identify trans-acting factors and cis-acting elements that might be important for conveying epigenetic memory through cell division, we isolated native (unfixed) chromosomes from metaphase-arrested cells using flow cytometry and performed LC-MS/MS to determine the repertoire of chromosome-bound proteins. Quantitative proteomic comparisons between metaphase-arrested cell lysates and chromosome-sorted samples revealed a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers (such as PRC2 and DNA methyl-transferases) and proteins governing chromosome architecture. We showed that deletion of PRC2, DNMT1/3a/3b or Mecp2 provoked an increase in the size of individual mitotic chromosomes consistent with de-condensation, as did experimental cleavage of cohesin complexes. These data provide a comprehensive inventory of chromosome-bound factors in pluripotent stem cells at mitosis and reveal an unexpected role for chromatin repressor complexes in preserving mitotic chromosome compaction.

Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.

Globally Vitamin D deficiency is a burning issue for the last two decades. Though the very high prevalence of hypovitaminosis D is worldwide even in the South-East Asia but there is very limited data in Bangladesh. This observational study was designed to assess the status of Vitamin D deficiency among the clinically apparent healthy population in the district of Jessore, Bangladesh from January 2017 to June 2017. Both the male and female clinically healthy participants between the age of 10 and 70 years residing more than 3 years in Jashore were included in this study, while those having renal or liver failure or any other chronic diseases and using drugs that could potentially interfere the metabolism of vitamin D were excluded from the study. Here we leveled serum 25(OH)D concentration of ≤20ngm/dl as deficient,20-30ngm/dl graded as insufficient and30ngm/dl graded as sufficient. Among 160 participants 43.1% (n=69) were male and 56.9% (n=91) were female. And 102(63.7%) participants had deficient vitamin D, 50(31.3%) had insufficient vitamin D and only 8(5%) had sufficient vitamin D. The mean serum 25(OH)D levels in our study was 18.60±6.59ngm/dl. The mean serum 25(OH)D level in case of male participants was 19.72±7.10ngm/dl whereas in female the corresponding figure was 17.74±6.07gm/dl and no significant difference observed among the man and women (p=0.059). Sun exposure play a vital role in the vitamin D deficiency and we observed only 1-2 hours/day sun exposure was sufficient for normal vitamin D level (p=0.001). In case of women who used veil had significantly lower level of vitamin D (p=0.001) due to lack of adequate sun exposure. Urban participants who also had significant low level of vitamin D due to the same reason (p=0.009). There was no deficiency observed in farmers and only 5(3.1%) had insufficient vitamin D. No significant difference observed in deficiency of vitamin D among the different age, sex, education level, skin complexion or BMI. But the obese participants had significant (p=0.041) lower vitamin D level. It was concluded with the high rate of vitamin D deficiency in this small scale study conducting in a district of Bangladesh demands a nationwide survey of vitamin D.

This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.

This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.

UV–Visible spectrophotometers have been used as a tool for chemical analysis over last few decades. The simplicity and ease of operation makes the instrument more reliable and convenient to use, and therefore, they find important application in many fields. In the present case, the performance evaluations of a UV–Visible spectrophotometer has been made for wavelength accuracy, stray light, resolution, photometric accuracy and photometric repeatability using Certified Reference Materials and other complimentary calibrators. The strategy adopted for the performance evaluation/calibration of the spectrophotometer were based on extensive use of different geo-standards of diverse matrices including the candidate geo-standards like Nephelinite, NKT-1, Syenite, CG-2, Limestone, ML-2 and River sediment, sdAR-1 studied recently as a part of 29th, 30th, 30Ath and 31st round of international geo-standards programmes held recently in addition to, employing a set practice of calibration being followed internationally.

A novel method for simple, rapid and effective separation of thorium for it’s determination in geological samples such as rocks, and minerals has been developed. Thorium forms 1:2 neutral complex with the bi-dentate ligand, 2,3-dihydroxynaphthalene (2,3-H2ND), which is extracted into ethylacetate at pH 5–6. Thorium from the organic phase is quantitatively stripped off using dilute hydrochloric acid, and then determined spectrophotometrically as well as by ICP-AES. The effects of variables affecting the quantitative extraction of thorium have been studied in detail. The method has been validated by analyzing certified reference materials (CRM). RSD of the method varied over the range 1 to 5 %. The method has been successfully applied to rocks and minerals.

The aim of the present paper is to develop a novel method for fluorimetric determination of uranium in rock/mineral solutions containing hydrolysable elements such as Nb, Ta, Zr and Ti sequestered by bi-fluoride. These rocks/minerals are decomposed with ammonium bi-fluoride (NH4HF2) and sulfuric acid (H2SO4) mixture. Uranium in such mineral solutions is selectively extracted into ethyl acetate with 2,3-dihydroxynaphthalene at pH 10–12 in the presence of cetyltrimethylammonium bromide, prior to its pellet fluorimetric determination. Optimizations of certain parameters such as the effects of fluoride fluxes, mineral acids, masking agents and diverse ions are discussed in detail. This method is applied for the determination of uranium in synthetic mixtures and a set of in-house reference refractory minerals including certified reference material X1807 with a high degree of accuracy and precision. The results for the refractory minerals using the proposed method are in excellent agreement with results obtained by other standard methods. The novelty of the proposed method is that the decomposition mixture (NH4HF2/H2SO4) inhibits the hydrolysis of hydrolysable elements by formation of their soluble fluoro complexes, and the separation of uranium using the complexing agent 2,3-dihydroxynaphthalene is more eco-friendly compared to existing the conventional solvent extraction system using aluminum nitrate as the salting out agent.

Melanin transfer from melanocytes to keratinocytes and subsequent accumulation in the supranuclear region is a critical process in skin pigmentation and protection against UVR. We have previously proposed that the main mode of transfer between melanocytes and keratinocytes is through exo/endocytosis of the melanosome core, termed melanocore. In this study, we developed an in vitro uptake assay using melanocores secreted by melanocytes. We show that the uptake of melanocores, but not melanosomes, by keratinocytes is protease-activated receptor-2–dependent. Furthermore, we found that the silencing of the early endocytic regulator Rab5b, but not the late endocytic regulators Rab7a or Rab9a, significantly impairs melanocore uptake by keratinocytes. After uptake, we observed that melanin accumulates in compartments that are positive for both early and late endocytic markers. We found that melanin does not localize to either highly degradative or acidic organelles, as assessed by LysoTracker and DQ-BSA staining, despite the abundance of these types of organelles within keratinocytes. Therefore, we propose that melanocore uptake leads to storage of melanin within keratinocytes in hybrid endocytic compartments that are not highly acidic or degradative. By avoiding lysosomal degradation, these specialized endosomes may allow melanin to persist within keratinocytes for long periods.

The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters in vitro. This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes.

The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin‐proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62‐dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross‐linked by the substrates. The reaction is inhibited by free ubiquitin, K48‐, and K63‐linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy.

Cryo-electron microscopy (cryo-EM) techniques have made a huge advancement recently, providing close to atomic resolution of the structure of protein complexes. Interestingly, this imaging technique can be performed in cells, giving access to the molecular machines in their natural context, therefore bridging structural and cell biology. However, in situ structural electron microscopy faces one major challenge, which is the ability to focus on specific subcellular regions to capture the objects of interest. Correlative light and electron microscopy (CLEM) is one very efficient solution for this. Here we present a sample preparation technique that enables cryo-sections of vitrified cell monolayers in an orientation that places the cryo-section parallel to the fluorescence imaging plane. The main advantage of this approach is that it exploits the potentials of CLEM for cryo-EM investigation, for selecting specific cells of interest in a heterogeneous population, or for finding identified subcellular regions on sections.

A relative reference measurement procedure for the determination of total mass fraction of uranium in mineralized rocks, ores and similar matrices using the application of a differential technique in inductively coupled plasma emission spectrometry at 409.014 nm wavelength is presented, based on the comparison of net reading or indication of reference ore solutions with a sample of similar but unknown mass fraction of uranium on the same sample weight or dilution basis. Under standard operating conditions of the instrument, ICP-OES measurements were performed at 409.014 nm wavelength after checking the instrument stability by C–Ar test, robustness of plasma and linearity response using aqueous standard U3O8 solutions. The estimated relative measurement uncertainty values obtained using the results of replicate analyses (“top-down” approach) in the ICP-OES procedure for mass fraction of uranium in low grade uranium ore IAEA samples and CANMET-reference uranium ore are S1, 0.0015; S2, 0.002; S3, 0.002; S4, 0.0015; and BL-2a, 0.001, respectively. Both approaches show low measurement uncertainty in the determination of total mass fraction of uranium without any chemical separation or extraction steps using a differential technique in the ICP-OES measurement procedure and are comparable with the differential technique in laser-induced fluorimetry (DT-LIF). Differential technique in spectrophotometry/laser fluorimetry/ICP-OES has inherent high metrological quality. Differential technique in the ICP-OES measurement procedure will be useful for the analysis of uranium in ores, certification of reference materials, borehole core assay, and other diverse applications in the nuclear fuel cycle.

A precise, accurate and rapid method for the sequential determination of FeO and Fe2O3 in rocks, soils and some non-refractory minerals by 1,10-phenanthroline spectrophotometry is described. Fe(II) and Fe(III) were leached from the sample (−200 mesh) using a mixture of NH4HF2 and H2SO4 at 40–80 °C for 10 min on a hot plate. Both Fe(II) and Fe(III) could be conveniently estimated sequentially from the same reaction mixture at the μg g−1 to percentage level. The method is better than the existing wet chemical methods, including the commonly used Pratt's titrimetric redox method, for Fe(II) and Fe(III) determinations in rock and soil samples in terms of precision, accuracy and rapidity. The throughput of the method was very high; at least forty to fifty samples could be estimated easily in a day. The results obtained compare favourably with those obtained by Pratt's method, as well as for certified/recommended values of a set of eleven certified reference materials having FeO and Fe2O3 contents in the range 0.21–14.63% and 0.58–8.48%, respectively. The optimised 1,10 phenanthroline method was found to be accurate to within 0.21% m/m FeO and 0.30% m/m Fe2O3 compared with the literature values of the certified reference materials studied.

Iron irrespective of its valency states forms bluish 1:1 cationic, bluish-violet 1:3 neutral and purple 1:3 anionic complexes with 2,3-dihydroxynaphthalene, at pH 2 - 3, 4 - 5 and 8 - 10, respectively. The cationic bluish complex is highly insensitive and not extractable in organic solvents. Similarly, the bluish violet 1:3 neutral complex is of moderate sensitivity, and it is extractable in organic solvents with reduced sensitivity. The purple 1:3 anionic complex is as such not extractable in any organic solvents. However, in the surfactant medium of the cationic surfactant, cetyltrimethylammonium bromide it is extractable in organic solvents with enhanced sensitivity. The 1:3 anionic complex which is extractable in organic solvents as an ion associate in the presence of cetyltrimethyl ammonium bromide is chosen for application to Silicate rocks, Minerals, Soils, Stream sediments, Concentrates, Tobacco leaves, Cigarettes and Waters, for accurate and sensitive determination of iron owing to facile extraction of iron, enhanced sensitivity high selectivity and better reproducibility of results. The present paper describes a systematic study on the development of an extraction spectrophotometric method for the determination of iron in samples of diverse matrices.

The metrological principle of ‘differential technique in laser-induced fluorimetry’ analysis is discussed and recommended as a reference measurement procedure for determination of total uranium content in ores and similar matrices. The estimated relative expanded uncertainty values obtained for uranium content in standard IAEA samples are, S 1, 0.04 g/kg, S 2, 0.06 g/kg, S 3, 0.04 g/kg, and for S 4, 0.10 g/kg, respectively. These low uncertainty values obtained for uranium show high metrological quality of differential technique. This reference measurement procedure guarantees the quality of an analytical result (accuracy, high precision, reliability, comparability, and traceability). Laser-induced fluorimetry will be useful for the analysis of uranium in ores, certification of reference materials, borehole core assay, and other diverse applications in nuclear fuel cycle. Differential technique in spectrophotometry/laser fluorimetry has inherent high metrological quality. In principle, laser-induced fluorimetry is an ideal technique for the very accurate determination of uranium by the use of appropriate fluorescence-enhancing reagents and methodology depending upon the concentration of uranium and sample matrices.

Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209–23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.

An extraction spectrophotometric method for iron determination in rocks, minerals, soils, stream sediments and water samples has been developed. At pH 3-4, iron (III) forms a 1:2:1 ternary complex with thiocyanate and cetyltrimethylammonium bromide (Fe/SCN/CTAB) which is extracted into ethyl acetate. The readily formed purple complex is suitable for extraction spectrophotometric determination of iron in rocks and related materials from submicrogram to milligram levels. The method is free from any interference due to commonly associated ions present in the matrices of rock samples. The present method is at least fourfold more sensitive ( e =3.2×10 4 l mol −1 cm −1 ) than the conventional thiocyanate method and, in addition to the enhanced sensitivity and selectivity, it has got definite advantages over the corresponding binary thiocyanate system in terms of substantial improvement in the stability of the complex formed and broadening of Beer's law adherence range (0–6.0 mg/l). The method has been applied to a number of geological and hydrogeochemical samples for the determination of iron and the results obtained have been found to be favourably comparable with those obtained from the standard methods.

2,3-Dihydroxynaphthalene forms a strong chelate with titanium over the pH range 4 to 9. At pH 4–5, titanium is extracted into ethyl acetate along with iron, leaving behind V, Nb, Mo, and a host of elements present in complex matrices of rock samples. In the extract, titanium is easily separated from iron after raising the pH of the medium and re-extracting. The method is free from any interference. The sensitivity of the method is 3.2 × 104 L · mol−1 cm−1. The method has been applied to a number of diverse samples including rocks and minerals. The precision of the Ti method is excellent. This method has been compared with tiron, chromotropic acid, diantipyrilmethane (DAM) and other existing spectrophotometric methods used in the analysis of rocks, ores and minerals. The proposed method has definite advantages over most spectrophotometric methods in terms of sensitivity, selectivity, reproducibility and simplicity.

The Rab27a GTPase regulates diverse processes involving lysosome-related organelles, including melanosome motility in melanocytes, and lytic granule release in cytotoxic T lymphocytes. Toward an understanding of Rab27a function, we searched for proteins that interact with Rab27a(GTP) using the yeast two-hybrid system and identified JFC1/Slp1, a protein of unknown function. JFC1/Slp1 and related proteins, including melanophilin, contain a conserved amino-terminal domain similar to the Rab3a-binding domain of Rabphilin-3. We used several methods to demonstrate that this conserved amino-terminal domain is a Rab27-binding domain. We show that this domain interacts directly, and in a GTP-dependent manner with Rab27a. Furthermore, overexpression of this domain in melanocytes results in perinuclear clustering of melanosomes, suggesting that this region is sufficient for interaction with, and perturbation of function of, Rab27a in a physiological context. Thus, we identified a novel family of Rab27-binding proteins. We also show that melanophilin associates with Rab27a and myosin Va on melanosomes in melanocytes, and present evidence that a domain within the carboxyl-terminal region of melanophilin interacts with the carboxyl-terminal tail of the melanocyte-specific splice isoform of myosin Va. Thus, melanophilin can associate simultaneously with activated Rab27a and myosin Va via distinct regions, and serve as a linker between these proteins.

PMID:25051489 WOS:000339551100063 The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the alpha 1 subunit of Na+, K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation. publishersversion published

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

A simple, rapid, cost-effective, accurate and precise differential laser-induced fluorimetric method for the direct determination of uranium in mineralised rocks at the percentage level is presented, based on the comparison of the fluorescence of the standard with a sample of similar but unknown concentration on the same sample weight basis, by the use of H 3 PO 4 –NH 4 H 2 PO 4 as a fluorescence enhancing reagent. The relative standard deviation of the method was 0.3–0.5% ( n =9) for 0.04 and 3.4% U 3 O 8 . The proposed technique is suitable for the determination of uranium in samples containing >0.01% uranium. The method has been evaluated using standards, SY-2, SY-3, low grade uranium ore-IAEA reference samples and core samples of diverse matrices. The results are in good agreement with the published data and those obtained by conventional fluorimetry and other methods and are of comparable precision to those obtained by titrimetric assay.

Regional Centre for Exploration and Research, AMDER/Department of Atomic Energy, Khasmahal, Jamshedpur-831 002, Jharkhand, India E-mail : ptarafder@sify.com A significant finding on the role of soluble organic matters, particularly orthodihydroxy phenols and naphthols in the suppression/inhibition of uranium fluorescence in water samples is reported. Most natural water samples do contain a number of soluble organic compounds having capacity to form chelates/ complexes with metal ions present in these waters. The organic load in natural water varies with the degree of pollution/eutrification of water bodies. Some of the soluble organic ligands such as phenolic compounds, amino acids, fulvic acids, etc. form so strong complexes with UO22+ ion that it cannot come out of the clutch of such complexes in order to form phosphate complex with fluorescence enhancing buffer such as fluran or pyrophosphoric acid used to measure uranium fluorescence. This phenomenon has been thoroughly verified/demonstrated in our laboratory by using a host of organic compounds, mostly phenols and naphthols which are expected to be present in natural water samples. Heavy suppression of uranium fluorescence has been observed with a number of such compounds. In line with this, suppression of uranium fluorescence in real water samples have also been observed. After destruction of the soluble organic compounds present in water samples, the actual fluorescence of uranium present in these waters could be successfully measured. Measurement of uranium fluorescence in most water samples is faulty unless a prior destruction of these soluble organic compounds is resorted to. &nbsp

Chemistry Laboratory, Regional Center for Exploration and Research, Atomic Minerals Directorate, Eastern Region, AMD Complex, Khasmahal, Jamshedpur-831 002, Jharkhand, India E-mail : ptarafder@sify.com National Metallurgical Laboratory, Burmamines, Jamshedpur-831 007, Jharkhand, India A simple and rapid separation procedure for the determination of total rare earth elements (lanthanides) including yttrium in geological samples such as rock, soil and sediments has been developed. Lanthanides form 1: 3 complex with O-O' type hi-dentate ligand, 2,3-dihydroxynaphthalene (2,3-H2ND) and are extracted in the ethyl acetate from aqueous solution at pH >8. Lanthanides from (lrganic phase are quantitatively and selectively stripped off at pH 6.3 ± 0.2 followed by its spectrophotometric determination using arsenazo-III. The effects of different experimental parameters such as pH of aqueous solution, concentration of 2,3-H2ND, electrolytes, equilibrium time, stripping agents, diverse ions etc. on the quantitative recoveries of lanthanides were studied in detail. The percentage recovery was checked by analyzing lanthanides in synthetic mixtures and it was found that more than 98% recovery of lanthanides was obtained by this method. The accuracy of the method was ascertained by analyzing some Certified Reference Materials (CRM) like syenite sample (SY/3) and gabro sample (MRG-1). The lanthanide values obtained for the CRMs were in excellent agreement with the recommended values, indicating that the method yields fairly accurate and the reproducible results as characterized by a relative standard deviation (RSD) of 1to 5% (n = 4). The method was successfully applied to silicate rock, soil and sediment samples. &nbsp

The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.

A sensitive and highly selective aqueous as well as extractive spectrophotometric method has been developed for the determination of titanium(IV) using 2,3-dihydroxynaphthalene (H(2)ND) as a chromogenic agent. The reagent (H(2)ND) forms a 1:3 (TiOH(3+):ligand) complex at pH 4-9. The molar absorptivity and Sandell's sensitivity are 3.2 x 10(4) l . mol(-1)mol . cm(-1) and 0.001 microg/cm(2), respectively at lambda(max) 375 nm. The method has been found highly selective for Ti(IV) determination in rock samples. Solvent extraction of Ti(IV) in ethylacetate greatly improves the detection limit of the method. The method has been successfully applied to diverse silicate rock samples and results obtained are favourably comparable with those obtained from the tiron method. The reagent (H(2)ND) used in the present investigation is a much better variant than tiron for titanium(IV) determination in silicate rock samples in terms of sensitivity, selectivity, operational simplicity and economy.

Synthesis of novel mixed complex fluoroberyllates of the types M2[BeF3(HC2O4)]·H2O (M = NH4 or Na), K2[BeF3(HC2O4)], M2[BeF3(NH2CH2COO)]·H2O (M = NH4 or Na), and M2[BeF2(H2PO4)2] (M = NH4 or K) has been achieved from the reaction of Be(OH)2 with MF and the corresponding co-ligands viz., oxalic acid, glycine, and orthophosphoric acid, at pH ca. 2. Vibrational spectroscopic evidences have been provided in respect of co-ordinated F− and the ligands HC2O4−, NH2CH2COO−, or H2PO4−. IR spectra of ammonium salt of the complexes demonstrate the presence of hydrogen bonding.

A method for the determination of chromium is presented, based on the oxidation of hydroxylamine hydrochloride to nitrous acid by chromium(VI) in acetic acid medium followed by diazotization of the nitrite produced with p -aminophenylmercaptoacetic acid and subsequent coupling of the diazonium salt with N -(1-napththyl)ethylenediamine dihydrochloride in acidic medium to form a stable blueish azo dye. The method is suitable for the determination of chromium(IV) from 0.04 to 1.2 mg 1 −1 in a 1.0-cm cuvette. The molar absorptivity and Sandell's sensitivity are 3.65 × 10 4 1 mol −1 cm −1 and 0.0014 μ cm −2 , respectively.

Summary Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis of cellular activation of MyoVa and MyoVIIa by Myrip. To answer these questions we used melanosome transport in skin melanocytes and RPE cells as read-outs of MyoVa and MyoVIIa activity. We found that Myrip recruits and activates MyoVa on skin melanosomes with similar efficiency to the established MyoVa activator Melanophilin (Mlph). Mutagenesis showed that a Myrip–Mlph conserved amphipathic helix (MMAH) is essential for MyoVa interaction while other Myrip regions, including the MyoVa exon F binding domain equivalent, play non-essential roles in this interaction. This suggests that, in contrast to Mlph, Myrip interacts with MyoVa lacking melanocyte-specific exon F. Parallel studies of RPE melanosome transport reveal that Myrip-specific inserts, but not the MMAH, are essential for MyoVIIa activation. We conclude that Myrip is a versatile Rab27a-associated myosin-activating protein that mediates cellular activation of MyoVa and MyoVIIa via non-overlapping domains.

Anti Phospholipid Syndrome (APS) is a relatively new conception of syndrome complex first noticed in 1983. It may be primary or secondary to other diseases like SLE, RA, Systemic sclerosis, behchet's syndrome, temporal arteritis, sjogren's syndrome psoriatic arthropathy etc. Clinical manifestations are consequences of vascular thrombosis and embolism like DVT, pulmonary embolism, stroke, TIA, complication of pregnancy with pregnancy loss. We report a 34 years married female housewife who presented with sudden onset of nausea, vomiting, vertigo, dysphagia, dysarthria and ataxia. She had a chronic leg ulcer. Neurological findings were consistent with lateral medullary syndrome due to stroke though she was normotensive, nondiabetic with normal lipid profile. She had history of two abortions in last three years. Investigations were done accordingly and she fulfilled the diagnostic criteria of APS. No secondary cause was detected after thorough clinical examination and laboratory investigations. She was treated symptomatically along with oral anticoagulation. She improved slowly but steadily.