The characterisation of a galactokinase from Streptomyces coelicolor

Promiscuous galactokinases (GalKs), which catalyse the ATP dependent phosphorylation of galactose in nature, have been widely exploited in biotechnology for the rapid synthesis of diverse sugar-1-phosphates. This work focuses on the characterisation of a bacterial GalK from Streptomyces coelicolor (ScGalK), which was overproduced in Escherichia coli and shown to phosphorylate galactose. ScGalK displayed a broad substrate tolerance, with activity towards Gal, GalN, Gal3D, GalNAc, Man and L-Ara. Most interestingly, ScGalK demonstrated a high activity over a broad pH and temperature range, suggesting that the enzyme could be highly amenable to multi-enzyme systems

Using Automated Glycan Assembly (AGA) for the Practical Synthesis of Heparan Sulfate Oligosaccharide Precursors

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography

Polyvalent Glycan-Quantum Dots as a Multifunctional Tool for Revealing Thermodynamic, Kinetic and Structural Details of Multivalent Lectin-Glycan Interactions

Multivalent lectin–glycan interactions (MLGIs) are widespread and vital for biology. Their binding biophysical and structural details are thus highly valuable, not only for the understanding of binding affinity and specificity mechanisms but also for guiding the design of multivalent therapeutics against specific MLGIs. However, effective techniques that can reveal all such details remain unavailable. We have recently developed polyvalent glycan quantum dots (glycan-QDs) as a new probe for MLGIs. Using a pair of closely related tetrameric viral-binding lectins, DC-SIGN and DC-SIGNR, as model examples, we have revealed and quantified their large affinity differences in glycan-QD binding are due to distinct binding modes: with simultaneous binding for DC-SIGN and cross-linking for DC-SIGNR. Herein, we further extend the capacity of the glycan-QD probes by investigating the correlation between binding mode and binding thermodynamics and kinetics and further probing a structural basis of their binding nature. We reveal that while both lectins’ binding with glycan-QDs is enthalpy driven with similar binding enthalpy changes, DC-SIGN pays a lower binding entropy penalty, resulting in a higher affinity than DC-SIGNR. We then show that DC-SIGN binding gives a single second-order kon rate, whereas DC-SIGNR gives a rapid initial binding followed by a much slower secondary interaction. We further identify a structural element in DC-SIGN, absent in DC-SIGNR, that plays an important role in maintaining DC-SIGN’s MLGI character. Its removal switches the binding from being enthalpically to entropically driven and gives mixed binding modes containing both simultaneous and cross-linking binding behavior, without markedly affecting the overall binding affinity and kinetics

Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host–pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein, we describe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors, enabling the enzymatic synthesis of Pse5Ac7Ac-containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late-stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot two-enzyme process using biosynthetic enzymes. Finally, screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac-terminated glycosides.</p

Glycan-Gold Nanoparticles as Multifunctional Probes for Multivalent Lectin-Carbohydrate Binding: Implications for Blocking Virus Infection and Nanoparticle Assembly

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin’s binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection

A comparative analysis of COVID-19 outbreak on age groups and both the sexes of population from India and other countries

INTRODUCTION: The study of epidemiological outcomes of COVID-19 in the affected countries needs to be conducted to implement an effective strategy. METHODOLOGY: Our study included age and sex-based analysis of epidemiological data of infected and deceased patients from various countries. The patient data was graphically depicted with the slope's calculation to describe a gradual or steep spread of the disease along with subsequent rise or fall in the death reports. RESULTS: Population groups of 20-49 years of age and 50 years-above were highly vulnerable to infection. Interestingly, 20-49 years of age group was most affected in India. However, higher population of the deceased were reported in the 50 years-above in all countries. India and South Korea demonstrated a gradual appearance of COVID-19 positive cases than other countries illustrated by reduced slope %. Further the highest percentage of infected people and deaths were reported from the densely populated states of India. We observed a sex independent prevalence of COVID-19. The BCG and JE vaccine are unique in the vaccination regime of India and South Korea. CONCLUSIONS: Reduced ACE-2 expression in the children's nasal epithelium may be responsible for reduced SARS-CoV-2 susceptibility. Countries showed varying patterns in COVID-19 spread and associated mortality. It may be influenced by factors, such as screening strategy, countries demography, implementation of lockdown, etc. Due to limited evidence, it would be difficult to point to the influence of the virus on either sexes. Although vaccines may stimulate non-specific immunity, experimental proofs are needed to demonstrate the potential of any vaccine against SARS-CoV-2

Probing Thermodynamics, Kinetics and Structural Details of Multivalent Lectin-Glycan Interactions by Quantum Dot-FRET

Multivalent lectin-glycan interactions (MLGIs) are widely employed for bio- recognition and discrimination, but they are also exploited by pathogens to infect host cells. Their biophysical details (e.g. thermodynamics, kinetics, binding modes and binding site orientation) are thus highly valuable, not only for elucidating the underlying mechanisms, but also for guiding the design of multivalent therapeutics against specific MLGIs. However, these details are not readily available due to the limitations of conventional biophysical techniques in probing such complex, flexible interactions. We have recently established densely glycosylated quantum dots (glycan-QDs) as novel structural probes for MLGIs. Using a pair of important, almost identical tetrameric lectins, DC-SIGN and DC-SIGNR, as the model lectins, we have shown that glycan-QDs can not only provide quantitative binding affinities but also dissect their distinct binding modes: DC-SIGN binds simultaneously with one glycan-QD whereas DC-SIGNR inter-cross-links. Herein, we further extend the capacity of the glycan-QD probes to investigate how binding mode affects the binding thermodynamics and kinetics, and probe a structural basis of their binding nature. We show that, while both lectin-glycan-QD interactions are enthalpy driven, with similar binding enthalpy changes (~4 times that of monovalent binding measured by ITC), DC-SIGN binding pays a lesser entropy penalty than DC-SIGNR, giving rise to a stronger affinity. We also reveal that a short C-terminal segment at the flexible junction between the tetramerization domain and glycan binding domain in DC-SIGN, absent in DC-SIGNR, plays a critical role in maintaining DC-SIGN’s glycan-QD binding properties: its removal leads to an entirely different binding enthalpy and entropy profile, despite maintaining the same binding mode. Furthermore, we show that the simultaneous lectin-glycan-QD binding partners give single 2nd-order kon rates which rapidly reach saturation, whereas cross-linking partners give two distinct on-rates: a rapid initial association step, followed by a much slower secondary interaction. Together, our work have established glycan-QDs as a powerful new biophysical platform for solution-based MLGI studies which can provide a wide range of important biophysical parameters

Outcome of minimally invasive surgery in the management of tuberculous spondylitis

Introduction: With the advancement of instrumentation and minimally access techniques in the field of spine surgery, good surgical decompression and instrumentation can be done for tuberculous spondylitis with known advantage of MIS (minimally invasive surgery). The aim of this study was to assess the outcome of the minimally invasive techniques in the surgical treatment of patients with tuberculous spondylodiscitis. Materials and Methods: 23 patients (Group A) with a mean age 38.2 years with single-level spondylodiscitis between T4-T11 treated with video-assisted thoracoscopic surgery (VATS) involving anterior debridement and fusion and 15 patients (Group B) with a mean age of 32.5 years who underwent minimally invasive posterior pedicle screw instrumentation and mini open posterolateral debridement and fusion were included in study. The study was conducted from Mar 2003 to Dec 2009 duration. The indication of surgery was progressive neurological deficit and/or instability. The patients were evaluated for blood loss, duration of surgery, VAS scores, improvement in kyphosis, and fusion status. Improvement in neurology was documented and functional outcome was judged by oswestry disability index (ODI). Results: The mean blood loss in Group A (VATS category) was 780 ml (330-1180 ml) and the operative time averaged was 228 min (102-330 min). The average preoperative kyphosis in Group A was 38° which was corrected to 30°. Twenty-two patients who underwent VATS had good fusion (Grade I and Grade II) with failure of fusion in one. Complications occurred in seven patients who underwent VATS. The mean blood loss was 625 ml (350-800 ml) with an average duration of surgery of 255 min (180-345 min) in the percutaneous posterior instrumentation group (Group B). The average preoperative segmental (kyphosis) Cobb′s angle of three patients with thoracic TB in Group B was 41.25° (28-48°), improved to 14.5°(11°- 21°) in the immediate postoperative period (71.8% correction). The average preoperative segmental kyphosis in another 12 patients in Group B with lumbar tuberculosis of 20.25° improved to -12.08° of lordosis with 32.33° average correction of deformity. Good fusion (Grade I and Grade II) was achieved in 14 patients and Grade III fusion in 1 patient in Group B. One patient suffered with pseudoarthrosis/doubtful fusion with screw loosening in the percutaneous group. Conclusion: Good fusion rate with encouraging functional results can be obtained in caries spine with minimally invasive techniques with all the major advantages of a minimally invasive procedures including reduction in approach-related morbidity

Integrating the issue of infrastructural investment with economic growth: The case of India

The development of a country’s infrastructure is instrumental in accelerating its economic growth. The inadequacy in the infrastructure provisions hinders population to promote self reliance in economic sectors, thereby proving to be a hindering factor to economic growth. Through this paper, we have aimed to investigate the relationship between investment in key infrastructure sectors and economic growth, in order to see how these sectors impact India’s economic growth and how significant this impact is. Further, a detailed qualitative analysis of all the infrastructure sectors involved in our study has been done with a focus on explaining the reasons behind significance/ insignificance of a particular sector. In the final part of analysis, a budget allocation model has been formulated with the help of linear programming technique. This model gives us a fresh viewpoint of the prospective inclination of government budget, and its extent of allocation to the diversity of infrastructure sectors