Biosynthesis of UDP-α-N-Acetyl-d-mannosaminuronic Acid and CMP-ß-N-Acetyl-d-neuraminic Acid for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in North America and Europe. The exterior surface of the bacterial cell wall is attached to a polymeric coat of sugar molecules known as the capsular polysaccharide (CPS) that helps protect the organism from the host immune response. The CPS is composed of a repeating sequence of common and unusual sugar residues. In the HS:11 serotype of C. jejuni, we identified two enzymes in the gene cluster for CPS formation that are utilized for the biosynthesis of UDP-α-N-acetyl-d-mannosaminuronic acid (UDP-ManNAcA). In the first step, UDP-α-N-acetyl-d-glucosamine (UDP-GlcNAc) is epimerized at C2 to form UDP-α-N-acetyl-d-mannosamine (UDP-ManNAc). This product is then oxidized by a NAD+-dependent C6-dehydrogenase to form UDP-ManNAcA. In the HS:6 serotype (C. jejuni strain 81116), we identified three enzymes that are required for the biosynthesis of CMP-ß-N-acetyl-d-neuraminic acid (CMP-Neu5Ac). In the first step, UDP-GlcNAc is epimerized at C2 and subsequently hydrolyzed to form N-acetyl-d-mannosamine (ManNAc) with the release of UDP. This product is then condensed with PEP by N-acetyl-d-neuraminate synthase to form N-acetyl-d-neuraminic acid (Neu5Ac). In the final step, CMP-N-acetyl-d-neuraminic acid synthase utilizes CTP to convert this product into CMP-Neu5Ac. A bioinformatic analysis of these five enzymes from C. jejuni serotypes HS:11 and HS:6 identified other bacterial species that can produce UDP-ManNAcA or CMP-Neu5Ac for CPS formation.

Biosynthesis of Cytidine Diphosphate-6-d-Glucitol for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in chickens and is the leading cause of human diarrheal disease worldwide. The various serotypes of C. jejuni produce structurally distinct capsular polysaccharides (CPSs) on the exterior surfaces of the cell wall. The capsular polysaccharide from C. jejuni serotype HS:5 is composed of a repeating sequence of d-glycero-d-manno-heptose and d-glucitol-6-phosphate. We previously defined the pathway for the production of d-glycero-d-manno-heptose in C. jejuni. Here, we elucidate the biosynthetic pathway for the assembly of cytidine diphosphate (CDP)-6-d-glucitol by the combined action of two previously uncharacterized enzymes. The first enzyme catalyzes the formation of CDP-6-d-fructose from cytidine triphosphate (CTP) and d-fructose-6-phosphate. The second enzyme reduces CDP-6-d-fructose with NADPH to generate CDP-6-d-glucitol. Using sequence similarity network (SSN) and genome neighborhood network (GNN) analyses, we predict that these pairs of proteins are responsible for the biosynthesis of CDP-6-d-glucitol and/or CDP-d-mannitol in the lipopolysaccharides (LPSs) and capsular polysaccharides in more than 200 other organisms. In addition, high resolution X-ray structures of the second enzyme are reported, which provide novel insight into the manner in which an open-chain nucleotide-linked sugar is harbored in an active site cleft.

Metabolic Pathways for the Biosynthesis of Heptoses Used in the Construction of Capsular Polysaccharides in the Human Pathogen Campylobacter jejuni.

Campylobacter jejuni is the leading cause of food poisoning in North America. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) that consists of a repeating sequence of 2-5 different carbohydrates that is anchored to the outer membrane. Heptoses of various configurations are among the most common monosaccharides that have been identified within the CPS. It is currently thought that all heptose variations derive from the modification of GDP-d-glycero-α-d-manno-heptose (GMH). From the associated gene clusters for CPS biosynthesis, we have identified 20 unique enzymes with different substrate profiles that are used by the various strains and serotypes of C. jejuni to make six different stereoisomers of GDP-6-deoxy-heptose, four stereoisomers of GDP-d-glycero-heptoses, and two stereoisomers of GDP-3,6-dideoxy-heptoses starting from d-sedoheptulose-7-phosphate. The modification enzymes include a C4-dehydrogenase, a C4,6-dehydratase, three C3- and/or C5-epimerases, a C3-dehydratase, eight C4-reductases, two pyranose/furanose mutases, and four enzymes for the formation of GMH from d-sedoheptulose-7-phosphate. We have mixed these enzymes in different combinations to make novel GDP-heptose modifications, including GDP-6-hydroxy-heptoses, GDP-3-deoxy-heptoses, and GDP-3,6-dideoxy-heptoses.

Biosynthesis of 3,6-Dideoxy-heptoses for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is the leading cause of food poisoning in the United States. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that helps protect the organism from the host immune system. The CPS is composed of a repeating sequence of common and unusual sugar residues, including relatively rare heptoses. In the HS:5 serotype, we identified four enzymes required for the biosynthesis of GDP-3,6-dideoxy-ß-l-ribo-heptose. In the first step, GDP-d-glycero-α-d-manno-heptose is dehydrated to form GDP-6-deoxy-4-keto-α-d-lyxo-heptose. This product is then dehydrated by a pyridoxal phosphate-dependent C3-dehydratase to form GDP-3,6-dideoxy-4-keto-α-d-threo-heptose before being epimerized at C5 to generate GDP-3,6-dideoxy-4-keto-ß-l-erythro-heptose. In the final step, a C4-reductase uses NADPH to convert this product to GDP-3,6-dideoxy-ß-l-ribo-heptose. These results are at variance with the previous report of 3,6-dideoxy-d-ribo-heptose in the CPS from serotype HS:5 of C. jejuni. We also demonstrated that GDP-3,6-dideoxy-ß-l-xylo-heptose is formed using the corresponding enzymes found in the gene cluster from serotype HS:11 of C. jejuni. The utilization of different C4-reductases from other serotypes of C. jejuni enabled the formation of GDP-3,6-dideoxy-α-d-arabino-heptose and GDP-3,6-dideoxy-α-d-lyxo-heptose.

Bifunctional Epimerase/Reductase Enzymes Facilitate the Modulation of 6-Deoxy-Heptoses Found in the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and the leading cause of food poisoning in the United States and Europe. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that consists of a repeating sequence of common and unusual carbohydrate segments. At least 10 different heptose sugars have thus far been identified in the various strains of C. jejuni. The accepted biosynthetic pathway for the construction of the 6-deoxy-heptoses begins with the 4,6-dehydration of GDP-d-glycero-d-manno-heptose by a dehydratase, followed by an epimerase that racemizes C3 and/or C5 of the product GDP-6-deoxy-4-keto-d-lyxo-heptose. In the final step, a C4-reductase catalyzes the NADPH reduction of the resulting 4-keto product. However, in some strains and serotypes of C. jejuni, there are two separate C4-reductases with different product specificities in the gene cluster for CPS formation. Five pairs of these tandem C4-reductases were isolated, and the catalytic properties were ascertained. In four out of five cases, one of the two C4-reductases is able to catalyze the isomerization of C3 and C5 of GDP-6-deoxy-4-keto-d-lyxo-heptose, in addition to the catalysis of the reduction of C4, thus bypassing the requirement for a separate C3/C5-isomerase. In each case, the 3'-end of the gene for the first C4-reductase contains a poly-G tract of 8-10 guanine residues that may be used to control the expression and/or catalytic activity of either C4-reductase. The three-dimensional structure of the C4-reductase from serotype HS:15, which only does a reduction of C4, was determined to 1.45 Å resolution in the presence of NADPH and GDP.

Product Specificity of C4-Reductases in the Biosynthesis of GDP-6-Deoxy-Heptoses during Capsular Polysaccharide Formation in Campylobacter jejuni.

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. A capsular polysaccharide that coats the exterior of the bacterium helps evade the host immune system. At least 33 different strains of C. jejuni have been identified, and the chemical structures of 12 different capsular polysaccharides (CPSs) have been characterized from various serotypes. Thus far, 10 different heptose sugars have been found in the chemically characterized CPSs, and each of these are currently thought to originate from the modification of GDP-d-glycero-d-manno-heptose by the successive action of 4,6-dehydratase (or C4-dehydrogenase), C3- or C3/C5-epimerase, and C4-reductase. Within the sequenced strains of C. jejuni, we have identified 25 different C4-reductases that cluster into nine groups at a sequence identity of >90%. Eight of the proteins from seven different clusters were purified, and their product profiles were determined with GDP-6-deoxy-4-keto-heptose substrates using NMR and ESI mass spectrometry. The isolated products included GDP-6-deoxy-l-gluco-heptose (serotype HS:2), GDP-6-deoxy-l-galacto-heptose (serotype HS:42), GDP-6-deoxy-l-gulo-heptose (serotype HS:15), GDP-6-deoxy-d-ido-heptose (serotypes HS:3, HS:4, and HS:33), GDP-6-deoxy-d-manno-heptose (serotype HS:53), and GDP-6-deoxy-d-altro-heptose (serotype HS:23/36). Based on these observations, the product specificity can be reliably predicted for 14 additional C4-reductases from C. jejuni. The remaining three C4-reductases are highly likely to be required for the biosynthesis of 3,6-dideoxy-heptose products.

C3- and C3/C5-Epimerases Required for the Biosynthesis of the Capsular Polysaccharides from Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and one of the leading causes of food poisoning in Europe and the United States. The outside of the bacterium is coated with a capsular polysaccharide that assists in the evasion of the host immune system. Many of the serotyped strains of C. jejuni contain a 6-deoxy-heptose moiety that is biosynthesized from GDP-d-glycero-d-manno-heptose by the successive actions of a 4,6-dehydratase, a C3/C5-epimerase, and a C4-reductase. We identified 18 different C3/C5-epimerases that could be clustered together into three groups at a sequence identity of >89%. Four of the enzymes from the largest cluster (from serotypes HS:3, HS:10, HS:23/36, and HS:41) were shown to only catalyze the epimerization at C3. Three enzymes from the second largest cluster (HS:2, HS:15, and HS:42) were shown to catalyze the epimerization at C3 and C5. Enzymes from the third cluster were not characterized. The three-dimensional structures of the epimerases from serotypes HS:3, HS:23/36, HS:15, and HS:41 were determined to resolutions of 1.5-1.9 Å. The overall subunit architecture places these enzymes into the diverse "cupin" superfamily. Within X-ray coordinate error, the immediate regions surrounding the active sites are identical, suggesting that factors extending farther out may influence product outcome. The X-ray crystal structures are consistent with His-67 and Tyr-134 acting as general acid/base catalysts for the epimerization of C3 and/or C5. Two amino acid changes (A76V/C136L) were enough to convert the C3-epimerase from serotype HS:3 to one that could now catalyze the epimerization at both C3 and C5.

Reaction Mechanism and Three-Dimensional Structure of GDP-d-glycero-α-d-manno-heptose 4,6-Dehydratase from Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in the United States and Europe. Surrounding the outside of the bacterium is a carbohydrate coat known as the capsular polysaccharide. Various strains of C. jejuni have different sequences of unusual sugars and an assortment of decorations. Many of the serotypes have heptoses with differing stereochemical arrangements at C2 through C6. One of the many common modifications is a 6-deoxy-heptose that is formed by dehydration of GDP-d-glycero-α-d-manno-heptose to GDP-6-deoxy-4-keto-d-lyxo-heptose via the action of the enzyme GDP-d-glycero-α-d-manno-heptose 4,6-dehydratase. Herein, we report the biochemical and structural characterization of this enzyme from C. jejuni 81-176 (serotype HS:23/36). The enzyme was purified to homogeneity, and its three-dimensional structure was determined to a resolution of 2.1 Å. Kinetic analyses suggest that the reaction mechanism proceeds through the formation of a 4-keto intermediate followed by the loss of water from C5/C6. Based on the three-dimensional structure, it is proposed that oxidation of C4 is assisted by proton transfer from the hydroxyl group to the phenolate of Tyr-159 and hydride transfer to the tightly bound NAD+ in the active site. Elimination of water at C5/C6 is most likely assisted by abstraction of the proton at C5 by Glu-136 and subsequent proton transfer to the hydroxyl at C6 via Ser-134 and Tyr-159. A bioinformatic analysis identified 19 additional 4,6-dehydratases from serotyped strains of C. jejuni that are 89-98% identical in the amino acid sequence, indicating that each of these strains should contain a 6-deoxy-heptose within their capsular polysaccharides.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/ß Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2ß2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and ß, to initiate, using its metallo-cofactor in ß2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(ß) and Y731(α) across the α/ß interface. Using 2,3,5-F3Y122-ß2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-ß2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor.

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.

Mechanism of copper incorporation in subunit II of cytochrome C oxidase from Thermus thermophilus: identification of intermediate species.

Detailed spectroscopic and kinetic studies of incorporation of copper ion in the wild type (WT) and the D111AA (AA = K, N, or E) mutants of the metal ion binding site of the soluble fragment of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) showed the formation of at least two distinct intermediates. The global analyses of the multiwavelength kinetic results suggested a four-step reaction scheme involving two distinct intermediates in the pathway of incorporation of copper ions into the apoprotein forming the purple dinuclear CuA. An early intermediate similar to the red copper binding proteins was detected in the WT as well as in all the mutants. The second intermediate was a green copper species in the case of WT TtCuA. Mutation of Asp111, however, formed a second intermediate that is distinctly different from that formed in the case of the WT protein, suggesting that mutants follow pathways of copper ion incorporation different from that in the WT protein. The electrostatic interaction between Asp111 and the coordinating His114 possibly plays a subtle role in the mechanism of incorporation of metal ion into the protein. The overall Kd for WT TtCuA was found to be ~8 nM, which changed with mutation of the Asp111 residue. The activation and thermodynamic parameters were also determined from the temperature- and pH-dependent multiwavelength kinetics, and the results are discussed to unravel the role of Asp111 in the mechanism of formation of the dinuclear CuA center in cytochrome c oxidase.

Role of the surface-exposed leucine 155 in the metal ion binding loop of the CuA domain of cytochrome c oxidase from Thermus thermophilus on the function and stability of the protein.

The role of Leu155 in the metal ion binding loop in the soluble CuA binding domain of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) was investigated by site-specific mutations of this residue to arginine (L155R) and glutamic acid (L155E). The UV-visible absorption and electron paramagnetic resonance spectra suggested that the Cu(2)S(2) core of TtCuA was almost unchanged by the mutations. The redox potential of the metal center in the L155R mutant was ~20 mV higher than that in the WT protein, while that of the L155E mutant was almost the same as that of the wild type (WT-TtCuA). The rate of transfer of an electron from cytochrome c(552) to the L155E mutant was much lower than that of transfer to the WT protein, while that for transfer to the L155R mutant was similar to that of WT-TtCuA. The total reorganization energy was increased for both the mutant proteins compared to WT-TtCuA. The results suggest that the presence of a negatively charged residue at the site of Leu155 in TtCuA possibly disfavors the protein-protein interaction between the two redox partners. The mutation also affected the equilibrium pH dependence of the protein. The thermal and thermodynamic stability of TtCuA was drastically decreased upon the mutation, which is most prominent in the L155R mutant. These studies indicate that the hydrophobic patch at the surface of TtCuA consisting of Leu155 is important for the transfer of an electron between cytochrome c(552) and TtCuA.

Role of steroid on management of limb swelling and local pain in haematotoxic snake bite.

Introduction: Local Pain and swelling in the biting area of haematotoxic snake bite victims are very common symptoms. This study was conducted to retrospectively observe the effect of oral Tab Prednisolone on a short-term basis as an add-on therapy of haematotoxic snake bite management in respect of Local Pain and swelling recovery. Materials and Methods: This retrospective descriptive study was conducted in a tertiary care hospital in West Bengal among 36 haematotoxic snake bite victims, admitted from February 2020 to January 2021. After collection of the data from hospital records and screened by inclusion and exclusion criteria, 36 participants were included in two groups based on the treatment regimen. Group A (n: 24) received only conventional treatment and Group B (n: 12) received oral Tab Prednisolone on a short-term basis as an add-on therapy of conventional treatment. Swelling was measured as a distance from the site of the bite with measuring tape in centimeters and pain was measured by a numerical rating pain scale (NRS) between 0 and 10. Ethical permission has been waived from the Institutional Ethical Review Committee. Result: A total of 36 patients (32 male and 4 female) were included in the study. Age (Mean ± SD) of the snake victim persons in Group A and Group B were 35.79 ± 8.34 years and 31.33 ± 6.47 years, respectively. The local swelling length and pain score reduced significantly among group B patients on day 6 in comparison to day 2. However, in Group A, the pain score and local swelling increased significantly on day 6 in comparison to day 2. Conclusion: A short course of systemic steroids as an adjuvant with anti-venom serum (ASV) for the management of local pain and edema in case of haematotoxic snake bite may be beneficial if there is no contraindication.

Tuning diamagnetic susceptibility of impurity doped quantum dots by noise-binding energy interplay.

In the present work we explore the role of noise-binding energy (BE) interplay on the diamagnetic susceptibility (DMS) of doped G a A s quantum dot (QD). Gaussian white noise has been invoked for the above exploration. And Gaussian impurity acts as a dopant in the present study. The mode of inclusion of noise to the system significantly affects the above interplay and thus prominently modulates the DMS profile. The outcomes of the inspection unfolds the way by which DMS of doped QD can be regulated by prudent adjustment of noise-BE interaction.

Influence of noise-binding energy interplay on DC-Kerr effect and electro-absorption coefficient of impurity doped quantum dots.

Present investigation focuses on analyzing the role of noise-binding energy (BE) interplay on the correction factors (CF) related to Kerr nonlinearity, DC-Kerr effect (DCKE) and electro-absorption coefficient (EAC) of GaAs quantum dot (QD) contaminated with impurity under the active presence of Gaussian white noise. The dopant impurity is modeled by a Gaussian potential. The noise-BE interplay does not give rise to any new interesting feature in case of CF from that in absence of noise. However, the said interplay prominently influences the DCKE and EAC profiles. This is justified by the emergence of distinct qualitative characteristics in the DCKE and EAC profiles that evidently depend on the mode of introduction of noise.

Influence of noise-binding energy interplay on the second and third-order nonlinear optical properties of impurity doped quantum dots.

We inspect the role of binding energy (BE) on second-order and third-order nonlinear optical (NLO) properties of doped G a A s quantum dot (QD). In the study ample stress is given on understanding the role of noise on the manifestations of these NLO properties. The profiles of these NLO properties are analyzed mainly on the basis of variation of two important criteria viz. peak-shift and peak-height as a function of BE. Both these features depend on the presence of noise, its pathway (mode) of introduction and sometimes on the identity of the NLO properties. The findings of the study deem significance in realizing the binding energy-dependence of the said NLO properties of low-dimensional semiconductor materials when noise contribution becomes noticeable.

Study of Vitamin B12 deficiency and peripheral neuropathy in metformin-treated early Type 2 diabetes mellitus.

BACKGROUND: Long-term therapy with metformin was shown to decrease the Vitamin B12 level and manifested as peripheral neuropathy. AIM: The aim of this study is to define the prevalence of Vitamin B12 deficiency in early Type 2 diabetic patients (duration ≤5 years or drug treatment ≤3 years) and the relationship among metformin exposure and levels of cobalamin (Cbl), folic acid, and homocysteine (Hcy) with severity of peripheral neuropathy. METHODOLOGY: This is a cross-sectional study involving randomly selected ninety patients (male 56, female 34) between age groups of 35 and 70 years, comparing those who had received >6 months of metformin (Group A) (n = 35) with those without metformin (Group B) (n = 35) and patients taking metformin with other oral hypoglycemic agent (Group C) (n = 20). Comparisons were made clinically, biochemically (serum Cbl, fasting Hcy, and folic acid), and with electrophysiological measures (nerve conduction studies of all four limbs). Comorbidities contributing to neuropathy were excluded from the study. RESULTS: Group A patients (54.28%) were prone to develop peripheral neuropathy comparing Group B (28.57%) and Group C (35%). There was significantly low plasma level of Cbl in Group A (mean 306.314 pg/ml) than in Group B (mean 627.543 pg/ml) and Group C (mean 419.920 pg/ml). There was insignificant low-level plasma folic acid in Group A (16.47 ng/ml) than in Group B (16.81 ng/ml) and Group C (22.50 ng/ml). There was significantly high level of Hcy in Group A (mean 17.35 µmol/L) and Group C (mean 16.99 µmol/L) than in Group B (mean 13.22 µmol/L). Metformin users even for 2 years showed evidence of neuropathy on nerve conduction velocity though their body mass index and postprandial blood sugar were maintained. There was significant difference in between groups regarding plasma Cbl, folic acid, and Hcy level as significance level <0.05 in all three groups (F [2, 87] = 28.1, P = 0.000), (F [2, 87] = 7.43, P = 0.001), (F [2, 87] = 9.76, P = 0.000). Post hoc study shows significant (P < 0.05) lowering of Cbl and Hcy level in Group A (mean = 306.314, standard deviation [SD] = 176.7) than in Group C (mean = 419.92, SD = 208.23) and Group B (mean = 627.543, SD = 168.33). DISCUSSION: Even short-term treatment with metformin causes a decrease in serum Cbl folic acid and increase in Hcy, which leads to peripheral neuropathy in Type 2 diabetes patients. A multicenter study with heterogeneous population would have increased the power of the study. We suggest prophylactic Vitamin B12 and folic acid supplementation or periodical assay in metformin user.

Prevalence of Skin Changes in Diabetes Mellitus and its Correlation with Internal Diseases: A Single Center Observational Study.

BACKGROUND AND AIM: This single-center observational cross-sectional study has been done in an attempt to find out the prevalence of various skin manifestations in diabetes patients (DM) and their correlation with diabetes control and complications. MATERIALS AND METHODS: Skin manifestations present over 12 months among those attend diabetes clinic were included in the study. Apart from demographic data and type, patients were also screened for micro vascular complications and control of diabetes over last 3 months. RESULTS AND DISCUSSION: Sixty (n = 60) diabetes patisents (Type 1 DM, 9 patients and Type 2 DM 51 patients) have been found to have various skin lesions. Thirty-one (51.67%) patients presented with infectious conditions, vascular complications were present in 21 (35%) and dermatomes belonging to the miscellaneous group were present in 50 (83.33%) patients. Pyoderma, diabetic dermopathy, and pruritus without skin lesions were found to be most common manifestations in infective, vascular and miscellaneous group, respectively. Higher level of HB1AC was found in patient with diabetic bulla (10.5 ± 0), scleredema (9.75 ± 0.77), lichen planus (9.3 ± 1.6), and acanthosis nigricans (9.15 ± 0.89). Patients with psoriasis and vitiligo had statistically significant lower level of glycosylated hemoglobin (P =< 0.001 and 0.03, respectively). However, no association of any kind of skin manifestation with DM with other microangiopathic complications was found in this study.

Correlation of intracranial atherosclerosis with carotid stenosis in ischemic stroke patients.

INTRODUCTION: Carotid stenosis is a major risk factor for ischemic stroke. However, the effect of carotid stenosis on the site of stroke is still under investigation. AIMS: This study aimed to elucidate how the presence of carotid stenosis influenced the pattern of stroke and also how it interacted with other risk factors for stroke. MATERIALS AND METHODS: Thirty-eight patients with ischemic stroke were included in this study and were investigated with carotid artery Doppler and magnetic resonance angiography for carotid stenosis and intracranial stenosis in the circle of Willis, respectively. Other known risk factors of stroke were also studied in and compared between the subgroups with and without carotid stenosis. RESULTS: In patients without carotid stenosis, anterior cerebral artery was the commonest site of stenosis. In patients with carotid stenosis, middle cerebral artery was the commonest site of stenosis. Overall, middle cerebral artery was the commonest territory of stroke. Patients with hypertension, diabetes and history of smoking had preferential stenosis of the anterior cerebral artery.