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BACKGROUND: Conventional B-mode breast ultrasonography, though the primary modality to determine benign or malignant nature of a solid breast lesion, sometimes encounters overlapping sonographic morphological features in a single lesion. Elastography leads to improvement by evaluating the structural aspects and characterization of the lesion as benign or malignant on the basis of multi-parametric assessment. OBJECTIVE: Determine the role of strain elastography (SE) and shear wave elastography (SWE) in differentiating benign and malignant breast lesions. DESIGN: Cross sectional SETTING: Radiology department of hospital PATIENTS AND METHODS: Patients meeting inclusion criteria referred to our hospital for ultrasonography followed by biopsy or surgical excisions were examined with B-mode ultrasonography and by both strain and shear wave elastography. MAIN OUTCOME MEASURES: Mean values of SE and SWE in benign and malignant breast lesions, determination of cutoff using AUC curves and sensitivity and specificity of both techniques. SAMPLE SIZE: One hundred breast lesions from 95 consecutive patients. RESULTS: The mean (SD) strain elastography ratio in the overall patient population was 4.1 (2.0). Cutoff for benign vs. malignant lesions was 2.86 on the ROC curve. The AUC was 0.911 (95%CI; 0.835-0.988: SE, 0.039) with a sensitivity of 95.8% and a specificity of 89.3%. For the SWE kPa values, the ROC curve showed the AUC was 0.929 (95% CI, 0.870-0.988; SE: 0.030, P

الملخص: أهداف البحث: أجريت هذه الدراسة لتحديد العوامل المرتبطة برجوع الورم المبكر بعد الجراحة لمرضى سرطان القولون والمستقيم الذين عولجوا بهدف الشفاء. طرق البحث: تم مراجعة جميع المرضى على التوالي الذين خضعوا للاستئصال العلاجي لسرطان القولون والمستقيم في الفترة من يناير ٢٠١٤ إلى ديسمبر ٢٠١٦. تلقى جميع المرضى إما العلاج الكيميائي المساعد أو تمت متابعتهم في معهد بانو لطب الأورام والعلاج الإشعاعي. ينتمي المرضى إلى المناطق الريفية في جنوب مقاطعة خيبر بختونخوا. النتائج: سجلنا ٧٢ مريضا؛ ٢٨ منهم لديه رجوع للورم بعد الجراحة في خلال السنتين الأولى (رجوع مبكر). في تحليل أحادي المتغير، وكان الانتكاس المبكر لمرضى سرطان القولون مرتبطا بشكل ملحوظ بالتقدم في السن أكثر من ٦٠ عاما. الحالة العقدية، المرحلة المرضية، عدد العقد التي استؤصلت والانتشار حول الشرج. باستخدام التحليل المتعدد المتغيرات، كان تجاوز العمر ٦٠ عاما واستئصال أقل من ١٢ عقدة ليمفاوية تنبؤات مستقلة للرجوع المبكر للورم. سجل الكبد الموقع الأكثر شيوعا للانتشار (٤٢.٨٪) في هذه الدراسة. الاستنتاجات: أظهرت الدراسة أن السن المتقدم واستئصال أقل من ١٢ من العقد الليمفاوية أثناء الجراحة هي تنبؤات كبيرة للرجوع المبكر بعد الجراحة. تحديد هؤلاء المرضى ذوي المخاطر العالية أثناء المتابعة بطرق علاجية مطورة يمكن أن يحسن البقاء على قيد الحياة بدون مرض. Abstract: Objectives: We conducted this study to determine the factors associated with early postoperative recurrence in colon cancer patients treated with curative intent. Methods: All consecutive patients who underwent curative resection for colon cancer between January 2014 and December 2016 were reviewed. All patients received either adjuvant chemotherapy or follow-up at the Bannu Institute of Nuclear Medicine Oncology and Radiotherapy (BINOR). The patients lived in rural areas of southern Khyber Pakhtunkhwa province. Results: We enrolled 72 patients, 28 of whom experienced a postoperative recurrence within 2 years (early recurrence). In univariate analysis, postoperative early relapse was significantly correlated with advanced age (>60 years, p = 0.030), nodal status (p = 0.012), pathological stage (p = 0.013), number of nodes removed (p

In colorectal carcinoma, carcinoembryonic antigen (CEA) is a recommended marker for surveillance after curative resection. The aim of the present study was to determine the association of preoperative CEA with recurrence of colorectal carcinoma in our population. The study included 55 patients with all operable stages of colorectal adenocarcinoma treated during the 2012-2014 period, evaluated retrospectively and followed-up for recurrence for 2 years. Data on the baseline (preoperative) CEA levels were retrieved from patient files. On data analysis, SPSS 16.0 was used. In patients with normal preoperative CEA, the rate of recurrence was significantly low (p=0.008) and the likelihood of no recurrence 1.55-fold greater as compared to patients with raised initial CEA levels (p=0.028). In patients with raised preoperative CEA, the risk of recurrence was 5.26-fold greater as compared to those with normal CEA levels (p=0.028). A significant weak positive correlation (rs=0.297) was found between raised CEA and recurrence. A highly significant (p=0.002) moderate positive correlation was recorded in patients aged

Objectives: Different clinicopathological and molecular features have been demonstrated between right and left sided colon cancers. We aimed to characterize colon cancer and sidedness among a North-Pakistani rural population diagnosed with colon cancer in our institution. Methods: Seventy patients were included in the study that received adjuvant chemotherapy at Bannu Institute of Nuclear Medicine Oncology and Radiotherapy) Bannu, Pakistan from January 2014 to December 2017. Chi-square test was used for significance of categorical variables. p-Values less than 0.05 were considered significant. Results: Mean age at diagnosis for right side colon cancer patients was 43.94 years and for left side colon cancer, it was 49.83 with no significant difference. Male patients were presented more with right (77% vs. 54%, p = 0.044) and females with predominantly left sided tumours i.e. (46% vs. 23%, p = 0.044). Right sided cancer tended to be more poorly differentiated (20% vs. 0%, p = 0.020). Mucinous adenocarcinoma was seen mostly in right sided colon cancer (37% vs. 3%, p ≤ 0.001). There were more locally advanced presentation of right side colon cancer with more node positive (83% vs. 60%, p = 0.025) and lymphovascular invasion (51% vs. 37%, p = 0.016). Sigmoid colon was the most common tumour subsite involved. Conclusion: Our study is the first report of colon cancer in a rural population in North-Pakistan. An earlier onset of tumours (44–50 years) was observed in comparison with global data. Resumo: Objetivo: Características clínico-patológicas e moleculares distintas foram observadas em tumores de cólon no lado direito ou esquerdo. O presente estudo teve como objetivo caracterizar o câncer de cólon e sua lateralidade em uma população rural norte-paquistanesa diagnosticada com câncer de cólon nesta instituição. Métodos: O estudo incluiu 70 pacientes que foram submetidos a quimioterapia adjuvante no Instituto Bannu de Medicina Nuclear Radioterapia Oncológica (BINOR), Bannu, Paquistão, entre janeiro de 2014 e dezembro de 2017. O teste qui-quadrado foi utilizado para mensurar a significância das variáveis categóricas. Valores de p menores que 0,05 foram considerados significativos. Resultados: A média de idade ao diagnóstico entre pacientes com câncer de cólon no lado direito foi de 43,94 anos e entre aqueles com câncer de cólon no lado esquerdo, 49,83, sem diferença significativa. Os pacientes do sexo masculino apresentaram mais tumores no lado direito (77% vs. 54%, p = 0,044) e as pacientes do sexo feminino apresentaram mais tumores no lado esquerdo (46% vs. 23%, p = 0,044). Tumores mal diferenciados foram mais comumente observados no lado direito (20% vs. 0%, p = 0,020). Adenocarcinoma mucinoso foi observado principalmente em casos de tumores no lado direito (37% vs. 3%, p ≤ 0,001). A apresentação local estava mais avançada em tumores de cólon no lado direito, com mais linfonodos positivos (83% vs. 60%, p = 0,025) e invasão linfovascular (51% vs. 37%, p = 0,016). O cólon sigmoide foi o sublocal mais comum. Conclusão: O presente estudo é o primeiro relato de câncer de cólon em uma população rural no norte do Paquistão. Em comparação com dados globais, observou-se um surgimento mais precoce dos tumores (44-50 anos). Keywords: Colon cancer sidedness, Left sided colon cancer, Right sided colon cancer, Clinicopathological, Rural, Palavras-chave: Lateralidade do câncer de cólon, Câncer de cólon no lado esquerdo, Câncer de cólon no lado direito, Clínico-patológico, Rural

Introduction: Various studies on corrosion of Orthodontic appliances and numerous factors aggravating this process have been discussed in the past. The diverse environment of the oral cavity favours the degradation of the metal alloys which is of concern to an Orthodontist due to the prolonged duration of these appliances in the mouth. Of the various factors affecting the biodegradation of the metals one of the factors is the diet and the dietary spices. Inspite of being an integral part of daily cuisine, the literature explaining their role in corrosion of the Orthodontic appliances is scarce. Aim: To assess and compare in vitro corrosion of Orthodontic metal brackets immersed in solutions of different spices and salt in artificial saliva. Materials and Methods: A total of 20 metal orthodontic brackets (AO) were divided into 10 groups containing artificial saliva, coriander (coriandrum sativum), turmeric (curcuma longa), black pepper (piper nigrum), red chilli (capsicum annuum) and Salt (sodium chloride). Electrochemical corrosion of the orthodontic brackets was carried out in corrosion cell. The corrosion current density (Icorr), corrosion potential (Ecorr) and pitting potential (Epit) rates were calculated using Tafel analysis and potentiodynamic data. Photographic images were captured from the metallurgical microscope. Results: The present study demonstrated that turmeric and coriander showed reduced corrosion whereas salt, red chilli and black pepper have been found to enhance it. Increased corrosion was seen in all groups containing salt. Surface analysis under metallurgical microscope showed increased pitting in red chilli solution group while less in coriander solution group. Conclusion: Different spices affect corrosion of metal brackets. Turmeric and coriander showed reduced corrosion whereas salt, red chillies and black pepper have been found to enhance it. Increased corrosion was seen with solutions containing salt.

Some alkyl and aryl derivatives of 9H-purine-6-thiol were synthesized and evaluated in vitro and in vivo for potential antithyroid effects. Spectrophotometric studies demonstrated 1:1 charge transfer complexation between iodine and these compounds with quite high values of the formation constants. The blood assays of rats treated with these compounds revealed significant antithyroid activity for almost all the compounds, which was further supported by a histological study of the thyroid tissues of the animals. These compounds are expected to provide less toxic alternative of the existing medicines as the sulfa group, which is known to be a cause of toxicity of many drugs, is blocked by alkyl/aryl substituents.

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The phenyl ring of the title compound, C12H10N4S, a purine derivative, is oriented at a dihedral angle of 76.65 (6)° with respect to the purine ring system. An intermolecular N—H...N hydrogen bonds stabilizes the crystal structure.

Despite a major threat to the public health in tropical and subtropical regions, dengue virus (DENV) infections are untreatable. Therefore, efforts are needed to investigate cost-effective therapeutic agents that could cure DENV infections in future. The NS2B-NS3 protease encoded by the genome of DENV is considered a critical target for the development of anti-dengue drugs. The objective of the current study was to find out a specific inhibitor of the NS2B-NS3 proteases from all four serotypes of DENV. To begin with, nine plant extracts with a medicinal history were evaluated for their role in inhibiting the NS2B-NS3 proteases by Fluorescence Resonance Energy Transfer (FRET) assay. Among the tested extracts, Punica granatum was found to be the most effective one. The metabolic profiling of this extract revealed the presence of several active compounds, including ellagic acid, punicalin and punicalagin, which are well-established antiviral agents. Further evaluation of IC 50 values of these three antiviral molecules revealed punicalagin as the most potent anti-NS2B-NS3 protease drug with IC 50 of 0.91 ± 0.10, 0.75 ± 0.05, 0.42 ± 0.03, 1.80 ± 0.16 µM against proteases from serotypes 1, 2, 3 and 4, respectively. The docking studies demonstrated that these compounds interacted at the active site of the enzyme, mainly with His and Ser residues. Molecular dynamics simulations analysis also showed the structural stability of the NS2B-NS3 proteases in the presence of punicalagin. In summary, this study concludes that the punicalagin can act as an effective inhibitor against NS2B-NS3 proteases from all four serotypes of DENV.Communicated by Ramaswamy H. Sarma.

Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi. styMdtM (locus name; STY4874) is a multidrug efflux transporter of the major facilitator superfamily expressed in S . Typhi. Functional assays identified several residues important for its transport activity. Here, we used an AlphaFold model to identify additional residues for analysis by mutagenesis. Mutation of peripheral residue Cys185 had no effect on the structure or function of the transporter. However, substitution of channel-lining residues Tyr29 and Tyr231 completely abolished transport function. Finally, mutation of Gln294, which faces peripheral helices of the transporter, resulted in the loss of transport of some substrates. Crystallization studies yielded diffraction data for the wild-type protein at 4.5 Å resolution and allowed the unit cell parameters to be established as a  =  b  = 64.3 Å, c  = 245.4 Å, α = β = γ = 90°, in space group P4. Our studies represent a further stepping stone towards a mechanistic understanding of the clinically important multidrug transporter styMdtM.Communicated by Ramaswamy H. Sarma.

In the current study, we have evaluated the protective efficacy of the 'insertion domain' which is commonly found in the capsid penton base protein of many adenoviruses. Using the 'insertion domain' of the penton base protein of a representative fowl adenovirus, fowl adenovirus serotype 4 (FAdV-4), we find that the 'insertion domain' can readily be expressed in a soluble form in the bacterial system, and can be purified in sufficient quantities through simple chromatographic methods. We demonstrate that the 'insertion domain', when employed as a subunit vaccine candidate, provides complete protection against hydropericardium syndrome, caused by FAdV-4, in chickens. The data presented here indicate that the protein, adjuvanted with Montanide™ ISA71 VG, provides complete protection in chickens against a lethal FAdV-4 challenge after administration of two doses (100 μg of the protein per dose) two weeks apart (the first dose at the 7th day of life and a booster dose at the age of 21 days). Furthermore, the purified protein can be stored at low temperatures without any observable loss in the protein integrity up to one year, tested so far. Due to the conserved nature of the 'insertion domain' across the penton base protein of fowl adenoviruses, it is suggested that homologous insertion domains could be employed as highly stable and cost-effective subunit vaccine candidates against infections caused by respective fowl adenoviruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Salmonellae are foodborne pathogens and the major cause of gastroenteritis in humans. Salmonellae express multidrug efflux transporters that play a key role in their drug resistance, which is becoming an increasing problem for therapeutic intervention. Despite their biomedical importance, the mechanisms underlying substrate transport by multidrug efflux transporters remain poorly understood. Here, we describe the first characterization of a multidrug transporter belonging to the major facilitator superfamily from the genus Salmonella. We show that several clinical Salmonella Typhi (S. Typhi) isolates constitutively express the styMdtM (STY4874) gene, which encodes a known multidrug-resistance (MDR) transporter. Guided by the structure of the Escherichia coli (E. coli) homolog, we studied two residues critical for substrate transport, Asp25 and Arg111. Mutation of Asp25 to glutamate did not affect the transport function of styMdtM, whereas mutation to alanine reduced its transport activity, suggesting that a negative charge at this position is critical for substrate translocation across the membrane. Substrate-affinity measurements by intrinsic fluorescence spectroscopy showed that the Asp25Ala mutant retained its capacity to bind substrate, albeit at a lower level. Mutation of Arg111 to alanine resulted in a decrease in secondary structure content of the transporter, and mutation to lysine completely destabilized the structure of the transporter. A homology model of styMdtM suggests that Arg111 is important for stabilizing the transmembrane domain by mediating necessary interactions between neighboring helices. Together, our studies provide new structural and mechanistic insights into the Salmonella MDR transporter styMdtM., (© 2021 Wiley Periodicals LLC.)

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic β cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant.

100K is an important scaffolding protein of adenoviruses including fowl adenovirus serotype 4 (FAdV-4) that causes inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) in poultry. 100K carries out the trimerization of the major capsid hexon protein of the virus for the generation of new virions inside the target host cells. Despite its critical role for FAdV-4, no structural study, in particular, has been conducted so far. Here, the overexpression of soluble 100K protein was successfully carried out in E. coli using various expression constructs and purification yield of 3mg per litre culture volume was obtained. Gel filtration chromatography suggested that 100K protein exists in trimeric form. Circular dichroism and Fourier transform infrared spectroscopy clearly reveal that 100K protein folds with a high content of α-helices. The 3-dimentional homology model of the 100K protein, refined with molecular dynamics tools also depicts higher α-helical content within the protein model. Moreover, overexpressed recombinant 100K protein could be used to differentiate vaccinated and FAdV-4 infected chickens on the basis of higher serum anti 100K antibody titres. Our work provides preliminary structural and functional results to study biological role of the 100K protein and for further investigations to develop 100K inhibitors to control IBH-HPS in poultry., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This white paper, prepared by members of the Cardiac Safety Research Consortium (CSRC), discusses important issues regarding scientific and clinical aspects of long-term electrocardiographic safety monitoring during clinical drug development. To promote multistakeholder discussion of this topic, a Cardiac Safety Research Consortium-sponsored Think Tank was held on 2 December 2015 at the American College of Cardiology's Heart House in Washington, DC. The goal of the Think Tank was to explore how and under what circumstances new and evolving ambulatory monitoring technologies could be used to improve and streamline drug development. This paper provides a detailed summary of discussions at the Think Tank: it does not represent regulatory guidance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Escherichia coli glutamate/aspartate-proton symporter GltP is a member of the Dicarboxylate/Amino Acid:Cation Symporter family of secondary active transport proteins. A range of computational, chemical, biochemical and biophysical methods characterised evolutionary relationships, structural features, substrate binding affinities and transport kinetics of wild-type and mutant forms of GltP. Sequence alignments and phylogenetic analysis revealed close homologies of GltP with human glutamate transporters involved in neurotransmission, neutral amino acid transporters and with the archaeal aspartate transporter GltPh. Topology predictions and comparisons with the crystal structure of GltPh were consistent with eight transmembrane-spanning α-helices and two hairpin re-entrant loops in GltP. Amplified expression of recombinant GltP with C-terminal affinity tags was achieved at 10% of total membrane protein in E. coli and purification to homogeneity with a yield of 0.8 mg/litre. Binding of substrates to GltP in native inner membranes and to purified protein solubilised in detergent was observed and quantified using solid-state NMR and fluorescence spectroscopy, respectively. A homology model of GltP docked with L-glutamate identified a putative binding site and residues predicted to interact with substrate. Sequence alignments identified further highly conserved residues predicted to have essential roles in GltP function. Residues were investigated by measuring transport activities, kinetics and response to thiol-specific reagents in 42 site-specific mutants compared with cysteine-less GltP (C256A) having an apparent affinity of initial rate transport (K m ) for 3 H-L-glutamate of 22.6 ± 5.5 μM in energised E. coli cells. This confirmed GltP residues involved in substrate binding and transport, especially in transmembrane helices VII and VIII.

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials., (Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. All rights reserved.)

Newcastle disease virus (NDV) is an infectious agent of a large variety of birds, including chicken, which poses a real threat to the agriculture industry. Matrix (M) proteins of NDV and many other viruses perform critical functions during viral assembly and budding from the host cell. M-proteins are well conserved and therefore are potential targets for antiviral therapies. To validate this, we expressed the NDV M-protein in its native form in Saccharomyces cerevisiae and in inclusion bodies in Escherichia coli. Proper refolding of the recombinant protein produced in E. coli was verified using circular dichroism and infrared spectroscopies and electron microscopy. Immunization of chickens with the NDV M-protein elicited significant serum antibody titers. However, the antibodies conferred little protection against the ND following lethal viral challenges. We conclude that the M-protein is not exposed on the surface of the host cell or the virus at any stage during its life cycle. We discuss how the conserved M-protein can further be exploited as an antiviral drug target.

Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ~0.1 to ~1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC50 increased to ~2.0 and > 10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.

Metal ion homeostasis is important for healthy cell function and is regulated by metal ion transporters and chaperones. To explore metal ion binding to membrane transport proteins we have used cadmium-113 as a solid state NMR probe of the Escherichia coli zinc exporter ZitB present in native membrane preparations. Competition experiments with other metal ions indicated that nickel and copper are also able to bind to this protein. Metal ion uptake studies were also performed using ZitB-reconstituted into proteoliposomes for a well established fluorescence assay. The results of both the solid state NMR and the uptake studies demonstrate that ZitB is potentially capable of transporting not only zinc but also cadmium, nickel and copper. The solid state NMR approach therefore offers great potential for defining the substrate spectrum of metal ion transporter proteins in their native membrane environments. Further, it should be useful for functional dissection of transporter mechanisms by facilitating the identification of functional residues by mutational studies.

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.

Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K(m) values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia.