Understanding the Acceptability of Broadly Neutralizing Antibodies for HIV Prevention Among At-Risk Populations and Feasibility Considerations for Product Introduction in India: Protocol for a Qualitative Study.

BACKGROUND: Acceptability and preference research play a crucial role in the design, evaluation, and implementation of any new prevention product in any geographical setting. They also play a critical role in the development of clinical guidelines and policies. A wide range of acceptability studies have been conducted in diverse general and key populations for various new HIV prevention products worldwide. As clinical development strategies are being developed for clinical studies of broadly neutralizing antibodies (bNAbs) as potential HIV prevention products, appropriately tailoring them to address the type of HIV epidemic at hand would be critical for efficient uptake within in-country public health systems and decrease adoption and adherence challenges. Accomplishing this will require comprehensive acceptability and feasibility studies to inform multisectoral efforts that increase access to these products and national policies supportive of access to health care for those in most need. Thus, it is both opportune and important to undertake focused efforts toward informing product development strategies. OBJECTIVE: This study aims to understand preferences for product attributes and key behavioral factors influencing adoption and uptake of bNAb prevention products among end-users including female sex workers, men who have sex with men, transgender women, people who inject drugs, and adolescent girls and young women in India and understand the key health system and programmatic perspectives toward the introduction of bNAb prevention products from health service providers and policy makers in India. METHODS: A multisite study will be conducted in Delhi, Mumbai, and Chennai to capture the differences in perspectives among diverse end-users and key informants across the country. The study will use a multimethods design using focus group discussions, in-depth interviews, simulated behavioral experiments, and key informant interviews. A total of 30 focus group discussions, 45 in-depth interviews, 15 simulated behavioral experiments sessions, and 15 key informant interviews will be conducted across 3 sites. RESULTS: The data collected and analyzed will enable insights on which specific product attributes matter the most to the populations and why some attributes are less preferred; contextual drivers of preferences and choices at individual, interpersonal, social, and structural levels; and relative positioning of bNAb products among other potential HIV prevention products. Insights from the health service providers and policy makers will provide a critical understanding of the need perception of the potential product in the existing product landscape and what additional efforts and resources are required for potential introduction, delivery, and uptake of the bNAb products in the Indian context. CONCLUSIONS: Insights generated from the abovementioned objectives will represent perspectives of populations of interest across geographies in India, will provide an overview of the acceptability of bNAb products and the feasibility of their introduction in this region, and will inform product development strategies. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/47700.

Engagement of vulnerable communities in HIV prevention research in India: a qualitative investigation.

BACKGROUND: Meaningful community engagement (CE) in HIV prevention research is crucial for successful and ethically robust study implementation. We conducted a qualitative study to understand the current CE practices in HIV prevention research and to identify expressed and implicit reasons behind translational gaps highlighted by communities and researchers. METHODS: For this exploratory qualitative study, we recruited a purposive sample of participants from Indian government-recognised key populations such as men who have sex with men, transgender women, people who inject drugs and female sex workers; general population adults and adolescents/youth; and researchers. We conducted 13 virtual focus groups (n = 86) between July and October 2021. Data were explored from a critical realist perspective and framing analysis (i.e., examining how the participants framed the narratives). RESULTS: Participants reported that study communities, especially those from key populations, were primarily involved in data collection, but not necessarily with optimal training. Involvement of communities before the start of the study (e.g., obtaining feedback on the study's purpose/design) or once the study is completed (e.g., sharing of findings) were highlighted as priorities for meaningful engagement. Participants suggested meaningful CE in all stages of the study: (1) before the study-to get inputs in finalising the study design, drafting comprehensible informed consent forms and culturally-appropriate data collection tools, and deciding on appropriate monetary compensation; (2) during the study-adequate training of community field research staff; and (3) after the study-sharing the draft findings to get community inputs, and involving communities in advocacy activities towards converting evidence into action, policy or programs. Timely and transparent communications with communities were explicitly stated as critical for gaining and maintaining trust. Mutual respect, reciprocity (e.g., appropriate monetary compensation) and robust community feedback mechanisms were considered critical for meaningful CE. CONCLUSIONS: The findings highlighted the translational gaps and priority areas for capacity building to strengthen CE in HIV prevention research. It is not only important to engage communities at various stages of research but to understand that trust, dignity, respect, and reciprocity are fundamentally preferred ways of meaningful community engagement.

Engaging communities in HIV prevention research enhances the rigour and impact of research. We sought to understand the current community engagement practices and to identify how communities preferred to get involved in research. We explored these topics with key and general populations and researchers, by conducting 13 focus group discussions with 86 participants. We found that there was limited involvement of communities before the start of the study and after its completion, although trained community members were involved in data collection. Participants strongly suggested that the community should be involved throughout­before initiation, during the study and after study completion. Participants' preferred ways of engaging communities reflected that mutual respect, reciprocity and transparent communications are critical for meaningful and successful community engagement.

Nickel Ferrite Nanoparticles as an Adsorbent for Immobilized Metal Affinity Chromatography of Proteins.

A simple method of preparing amorphous nickel ferrite nanoparticles of about 5 nm diameter is described. These particles were characterized by dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). The nanoparticles were evaluated for their use as a magnetic material for immobilized metal affinity chromatography (IMAC). The ferrite nanoparticles bound to bovine serum albumin (BSA) and the binding fitted Langmuir isotherm model. A high capacity of 916 mg BSA/g dried nanoparticle was observed. Six proteins (Soybean trypsin inhibitor (STI), lactate dehydrogenase (LDH), papain, catalase, ß-galactosidase and casein) were used and all were found to bind at >90% level (except papain which showed 84% binding). All the proteins except LDH and ß-galactosidase could be eluted with 1 M imidazole and with % activity recovery of >80%. Papain could be purified from its dried crude latex by 5-fold and purified papain showed a single band on SDS-PAGE. These nanoparticles constitute a high capacity and are magnetic material useful for IMAC and do not require any pre-functionalization.

Evaluation of antiviral T cell responses and TSCM cells in volunteers enrolled in a phase I HIV-1 subtype C prophylactic vaccine trial in India.

T cells play an important role in controlling viral replication during HIV infection. An effective vaccine should, therefore, lead to the induction of a strong and early viral-specific CD8+ T cell response. While polyfunctional T cell responses are thought to be important contributors to the antiviral response, there is evidence to show that polyfunctional HIV- specific CD8+ T cells are just a small fraction of the total HIV-specific CD8+ T cells and may be absent in many individuals who control HIV replication, suggesting that other HIV-1 specific CD8+ effector T cell subsets may be key players in HIV control. Stem cell-like memory T cells (TSCM) are a subset of T cells with a long half-life and self-renewal capacity. They serve as key reservoirs for HIV and contribute a significant barrier to HIV eradication. The present study evaluated vaccine-induced antiviral responses and TSCM cells in volunteers vaccinated with a subtype C prophylactic HIV-1 vaccine candidate administered in a prime-boost regimen. We found that ADVAX DNA prime followed by MVA boost induced significantly more peripheral CD8+ TSCM cells and higher levels of CD8+ T cell-mediated inhibition of replication of different HIV-1 clades as compared to MVA alone and placebo. These findings are novel and provide encouraging evidence to demonstrate the induction of TSCM and cytotoxic immune responses by a subtype C HIV-1 prophylactic vaccine administered using a prime-boost strategy.

Association of circulatory Tfh-like cells with neutralizing antibody responses among chronic HIV-1 subtype C infected long-term nonprogressors and progressors.

HIV-1 vaccine functioning relies on successful induction of broadly neutralizing antibodies (bNAbs). CXCR3- circulatory T-follicular helper (cTfh) cells are necessary for inducing B-cells for generating bNAbs. Recent studies have suggested that CXCR3+ Tfh cells might also influence bNAb production. Plasma samples from 34 ART-Naïve HIV-1 infected individuals [long-term nonprogressors (LTNP)-19; Progressors-13] were tested against a heterologous virus panel (n = 11) from subtypes A, B, C, G, AC, BC and AE. Frequencies of CXCR3+ and CXCR3- cTfh-like cells in peripheral circulation were studied using flow cytometry. LTNP showed significantly lower CXCR3+ and higher CXCR3- cTfh-like cell frequencies, while neutralization breadth was observed to be broader in progressors. A positive correlation was observed between bNAb breadth and potency with CXCR3+PD-1+ cTfh-like cells in LTNP. Based on neutralization breadth, 9 HIV-1 infected individuals were classified as 'top neutralizers' and 23 as 'low neutralizers' and they did not show any correlations with CXCR3+ and CXCR3- cTfh-like cells. These preliminary data suggest that CXCR3+ similar to CXCR3- might possess significant functional properties for driving B-cells to produce bNAbs. Hence, an HIV vaccine which is capable of optimal induction of CXCR3+ cTfh cells at germinal centers might confer superior protection against HIV.

Induction and maintenance of bi-functional (IFN-γ + IL-2+ and IL-2+ TNF-α+) T cell responses by DNA prime MVA boosted subtype C prophylactic vaccine tested in a Phase I trial in India.

Effective vaccine design relies on accurate knowledge of protection against a pathogen, so as to be able to induce relevant and effective protective responses against it. An ideal Human Immunodeficiency virus (HIV) vaccine should induce humoral as well as cellular immune responses to prevent initial infection of host cells or limit early events of viral dissemination. A Phase I HIV-1 prophylactic vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009.The trial tested a HIV-1 subtype C vaccine in a prime-boost regimen, comprising of a DNA prime (ADVAX) and Modified Vaccine Ankara (MVA) (TBC-M4) boost. The trial reported that the vaccine regimen was safe, well tolerated, and resulted in enhancement of HIV-specific immune responses. However, preliminary immunological studies were limited to vaccine-induced IFN-γ responses against the Env and Gag peptides. The present study is a retrospective study to characterize in detail the nature of the vaccine-induced cell mediated immune responses among volunteers, using Peripheral Blood Mononuclear Cells (PBMC) that were archived during the trial. ELISpot was used to measure IFN-γ responses and polyfunctional T cells were analyzed by intracellular multicolor flow cytometry. It was observed that DNA priming and MVA boosting induced Env and Gag specific bi-functional and multi-functional CD4+ and CD8+ T cells expressing IFN-γ, TNF-α and IL-2. The heterologous prime-boost regimen appeared to be slightly superior to the homologous prime-boost regimen in inducing favorable cell mediated immune responses. These results suggest that an in-depth analysis of vaccine-induced cellular immune response can aid in the identification of correlates of an effective immunogenic response, and inform future design of HIV vaccines.

Performance analysis of a high-sensitivity multi-pinhole cardiac SPECT system with hemi-ellipsoid detectors.

PURPOSE: Single-photon emission computed tomography (SPECT) is a noninvasive imaging modality, used in myocardial perfusion imaging. The challenges facing the majority of clinical SPECT systems are low sensitivity, poor resolution, and the relatively high radiation dose to the patient. New generation systems (GE Discovery, DSPECT) dedicated to cardiac imaging improve sensitivity by a factor of 5-8. This improvement can be used to decrease acquisition time and/or dose. However, in the case of ultra-low dose (~3 mCi) injections, acquisition times are still significantly long, taking 10-12 min. The purpose of this work is to investigate a new gamma camera design with 21 hemi-ellipsoid detectors each with a pinhole collimator for cardiac SPECT for further improvement in sensitivity and resolution and reduced patient exposures and imaging times. METHODS: To evaluate the resolution of our hemi-ellipsoid system, GATE Monte-Carlo simulations were performed on point-sources, rod-sources, and NCAT phantoms. For average full-width-half-maximum (FWHM) equivalence with base flat-detector, the pinhole-diameter for the curved hemi-ellipsoid detector was found to be 8.68 mm, an operating pinhole-diameter nominally expected to be ~3 times more sensitive than state-of-the-art systems. Rod-sources equally spaced within the region of interest were acquired with a 21-detector system and reconstructed with our multi-pinhole (MPH) iterative OSEM algorithm with collimator resolution recovery. The results were compared with the results of a state-of-the-art system (GE Discovery) available in the literature. The system was also evaluated using the mathematical anthropomorphic NCAT (NURBS-based Cardiac Torso; Segars et al. IEEE Trans Nucl Sci. 1999;46:503-506) phantom with a full (clinical)-dose acquisition (25 mCi) for 2 min and an ultra-low dose acquisition of 3 mCi for 5.44 min. The estimated left ventricle (LV) counts were compared with the available literature on a state-of-the-art system (DSPECT). FWHM of the LV wall on MPH-OSEM-reconstructed images with collimator resolution recovery was estimated. RESULTS: On acquired rod-sources, the average resolution (FWHM) after reconstruction with resolution recovery in the entire region of interest (ROI) for cardiac imaging was on the average 4.44 mm (±2.84), compared to 6.9 mm (±1 mm) reported for GE Discovery (Kennedy et al., J Nucl Cardiol. 2014:21:443-452). For NCAT studies, improved sensitivity allowed a full-dose (25 mCi) 2-min acquisition (Ell8.68mmFD) which yielded 3.79 M LV counts. This is ~3.35 times higher compared to 1.13 M LV counts acquired in 2 min for clinical full dose for state-of-the-art DSPECT. The increased sensitivity also allowed an ultra-low dose acquisition protocol (Ell8.68 mmULD), 3 mCi (eight times less injected dose) in 5.44 min. This ultra-low dose protocol yielded ~1.23 M LV counts which was comparable to the full-dose 2-min acquisition for DSPECT. The estimated NCAT average FWHM at the LV wall after 12 iterations of the OSEM reconstruction was 4.95 and 5.66 mm around the mid-short-axis slices for Ell8.68mmFD and Ell8.68mmULD, respectively. CONCLUSION: Our Monte-Carlo simulation studies and reconstruction suggest using (inverted wineglass sized) hemi-ellipsoid detectors with pinhole collimators can increase the sensitivity ~3.35 times over the new generation of dedicated cardiac SPECT systems, while also improving the reconstructed resolution for rod-sources with an average of 4.44 mm in region of interest. The extra sensitivity may be used for ultra-low dose imaging (3 mCi) at ~5.44 min for comparable clinical counts as state-of-the-art systems.

Induction of circulating T follicular helper cells and regulatory T cells correlating with HIV-1 gp120 variable loop antibodies by a subtype C prophylactic vaccine tested in a Phase I trial in India.

A Phase I HIV-1 vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009 to test a subtype C prophylactic vaccine in a prime-boost regimen comprising of a DNA prime (ADVAX) and MVA (TBC-M4) boost. The trial demonstrated that the regimen was safe and well tolerated and resulted in enhancement of HIV-specific immune responses. Preliminary observations on vaccine-induced immune responses were limited to analysis of neutralizing antibodies and IFN-γ ELISPOT response. The present study involves a more detailed analysis of the nature of the vaccine-induced humoral immune response using specimens that were archived from the volunteers at the time of the trial. Interestingly, we found vaccine induced production of V1/V2 and V3 region-specific antibodies in a significant proportion of vaccinees. Variable region antibody levels correlated directly with the frequency of circulating T follicular helper cells (Tfh) and regulatory T cells (Treg). Our findings provide encouraging evidence to demonstrate the immunogenicity of the tested vaccine. Better insights into vaccine-induced immune responses can aid in informing future design of a successfulHIV-1 vaccine.

Broad neutralization response in a subset of HIV-1 subtype C-infected viraemic non-progressors from southern India.

Broadly neutralizing antibodies (bnAbs) have been considered to be potent therapeutic tools and potential vaccine candidates to enable protection against various clades of human immunodeficiency virus (HIV). The generation of bnAbs has been associated with enhanced exposure to antigen, high viral load and low CD4+ T cell counts, among other factors. However, only limited data are available on the generation of bnAbs in viraemic non-progressors that demonstrate moderate to high viraemia. Further, since HIV-1 subtype C viruses account for more than 50 % of global HIV infections, the identification of bnAbs with novel specificities is crucial to enable the development of potent tools to aid in HIV therapy and prevention. In the present study, we analysed and compared the neutralization potential of responses in 70 plasma samples isolated from ART-naïve HIV-1 subtype C-infected individuals with various disease progression profiles against a panel of 30 pseudoviruses. Among the seven samples that exhibited a neutralization breadth of ≥70 %, four were identified as 'elite neutralizers', and three of these were from viraemic non-progressors while the fourth was from a typical progressor. Analysis of the neutralization specificities revealed that none of the four elite neutralizers were reactive to epitopes in the membrane proximal external region (MPER), CD4-binding site and V1V2 or V3 glycan. However, two of the four elite neutralizers exhibited enhanced sensitivity towards viruses lacking N332 glycan, indicating high neutralization potency. Overall, our findings indicate that the identification of potent neutralization responses with distinct epitope specificities is possible from the as yet unexplored Indian population, which has a high prevalence of HIV-1 subtype C infection.

Simulations of a Multi-Pinhole SPECT Collimator for Clinical Dopamine Transporter (DAT) Imaging.

SPECT imaging of the dopamine transporter (DAT) is used for diagnosis and monitoring progression of Parkinson's Disease (PD), and differentiation of PD from other neurological disorders. The diagnosis is based on the DAT binding in the caudate and putamen structures in the striatum. We previously proposed a relatively inexpensive method to improve the detection and quantification of these structures for dual-head SPECT by replacing one of the fan-beam collimators with a specially designed multi-pinhole (MPH) collimator. In this work, we developed a realistic model of the proposed MPH system using the GATE simulation package and verified the geometry with an analytic simulator. Point source projections from these simulations closely matched confirming the accuracy of the pinhole geometries. The reconstruction of a hot-rod phantom showed that 4.8 mm resolution is achievable. The reconstructions of the XCAT brain phantom showed clear separation of the putamen and caudate, which is expected to improve the quantification of DAT imaging and PD diagnosis. Using this GATE model, point spread functions modeling physical factors will be generated for use in reconstruction. Also, further improvements in geometry are being investigated to increase the sensitivity of this base system while maintaining a target spatial resolution of 4.5-5 mm.

Protein aggregates: Forms, functions and applications.

Protein aggregation is implicated in diverse biochemical phenomena which include formation of inclusion bodies and amyloids. In recent years, inclusion bodies of many enzymes have been found to be catalytically active. Enzyme precipitates and their crystalline aggregates have found extensive applications in Biocatalysis in low water media. Protein aggregates also play a useful role in processed food. Enzymes are incorporated in detergents in the form of granulates. This review also looks at the various techniques which are used for characterizing protein aggregates.

Cross-Linked Enzyme Aggregates for Applications in Aqueous and Nonaqueous Media.

Extensive cross-linking of a precipitate of a protein by a cross-linking reagent (glutaraldehyde has been most commonly used) creates an insoluble enzyme preparation called cross-linked enzyme aggregates (CLEAs). CLEAs show high stability and performance in conventional aqueous as well as nonaqueous media. These are also stable at fairly high temperatures. CLEAs with more than one kind of enzyme activity can be prepared, and such CLEAs are called combi-CLEAs or multipurpose CLEAs. Extent of cross-linking often influences their morphology, stability, activity, and enantioselectivity.

Protein-Coated Microcrystals, Combi-Protein-Coated Microcrystals, and Cross-Linked Protein-Coated Microcrystals of Enzymes for Use in Low-Water Media.

Protein-coated microcrystals (PCMC) are a high-activity preparation of enzymes for use in low-water media. The protocols for the preparation of PCMCs of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. The combi-PCMC concept is useful both for cascade and non-cascade reactions. It can also be beneficial to combine two different specificities of a lipase when the substrate requires it. Combi-PCMC of CALB and Palatase used for the conversion of coffee oil present in spent coffee grounds to biodiesel is described. Cross-linked protein-coated microcrystals (CL-PCMC) in some cases can give better results than PCMC. Protocols for the CLPCMC of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. A discussion of their applications is also provided.

Design of a Multi-Pinhole Collimator for I-123 DaTscan Imaging on Dual-Headed SPECT Systems in Combination with a Fan-Beam Collimator.

For the 2011 FDA approved Parkinson's Disease (PD) SPECT imaging agent I-123 labeled DaTscan, the volume of interest (VOI) is the interior portion of the brain. However imaging of the occipital lobe is also required with PD for calculation of the striatal binding ratio (SBR), a parameter of significance in early diagnosis, differentiation of PD from other disorders with similar clinical presentations, and monitoring progression. Thus we propose the usage of a combination of a multi-pinhole (MPH) collimator on one head of the SPECT system and a fan-beam on the other. The MPH would be designed to provide high resolution and sensitivity for imaging of the interior portion of the brain. The fan-beam collimator would provide lower resolution but complete sampling of the brain addressing data sufficiency and allowing a volume-of-interest to be defined over the occipital lobe for calculation of SBR's. Herein we focus on the design of the MPH component of the combined system. Combined reconstruction will be addressed in a subsequent publication. An analysis of 46 clinical DaTscan studies was performed to provide information to define the VOI, and design of a MPH collimator to image this VOI. The system spatial resolution for the MPH was set to 4.7 mm, which is comparable to that of clinical PET systems, and significantly smaller than that of fan-beam collimators employed in SPECT. With this set, we compared system sensitivities for three aperture array designs, and selected the 3 × 3 array due to it being the highest of the three. The combined sensitivity of the apertures for it was similar to that of an ultra-high resolution fan-beam (LEUHRF) collimator, but smaller than that of a high-resolution fan-beam collimator (LEHRF). On the basis of these results we propose the further exploration of this design through simulations, and the development of combined MPH and fan-beam reconstruction.

Adding an appropriate amino acid during crosslinking results in more stable crosslinked enzyme aggregates.

Carrier free immobilization, especially crosslinked enzyme aggregates (CLEAs), has become an important design for biocatalysis in several areas. Adding amino acids during formation of CLEAs was found to give biocatalysts more stable at 55 °C and in the presence of 60% acetonitrile. The half-lives of CLEAs prepared with and without Arg addition were 21 and 15 h (subtilisin) and 4 and 1.6 h (α-chymotrypsin) at 55 °C, respectively. The corresponding half-lives during acetonitrile presence were 4.1 and 3.0 h (subtilisin) and 39 and 22 min (α-chymotrypsin), respectively. CLEAs made with Arg had higher percentages of alpha helix. CLEAs made by adding Lys, Ala, or Asp also were more stable. In the case of Thermomyces lanuginosus lipase (TLL), CLEA with Ala was even more stable than CLEA with Arg. The addition of a suitable amino acid, thus, enhances CLEA stabilities. The results are discussed in the light of earlier results on chemical modification of proteins and the observation that the Arg/Lys ratio is invariably high in the case of enzymes from thermophiles.

Lipase coated clusters of iron oxide nanoparticles for biodiesel synthesis in a solvent free medium.

Methyl or ethyl esters of long chain fatty acids are called biodiesel. Biodiesel is synthesized by the alcoholysis of oils/fats. In this work, lipase from Thermomyces lanuginosus was precipitated over the clusters of Fe3O4 nanoparticles. This biocatalyst preparation was used for obtaining biodiesel from soybean oil. After optimization of both immobilization conditions and process parameters, complete conversion to biodiesel was obtained in 3h and on lowering the enzyme amount, as little as 1.7U of lipase gave 96% conversion in 7h. The solvent free media with oil:ethanol (w/w) of 1:4 and 40°C with 2% (w/w) water along with 20% (w/w) silica (for facilitating acyl migration) were employed for reaching this high % of conversion. The biocatalyst design enables one to use a rather small amount of lipase. This should help in switching over to a biobased production of biodiesel.

Dual bioimprinting of Thermomyces lanuginosus lipase for synthesis of biodiesel.

Use of biodiesel as an alternative to non-renewable sources of energy has become an attractive option in recent years. The enzymatic synthesis of biodiesel by transesterification of fats/oils with an alcohol is a much more sustainable route than the chemical method. However, cost effectiveness of the enzymatic route is a major barrier in its commercialization. In this work, a high activity biocatalyst design of Thermomyces lanuginosus lipase is made by dually bioimprinting it with substrate and a surfactant (which is believed to open up the lid covering the active site of the lipase) during precipitation of the lipase in organic solvent. When the lipase was bioimprinted with only the surfactants, 28 U of the enzyme/g of oil could yield 99% biodiesel from soybean oil in about 4 h. However, when dually bioimprinted even very low enzyme load 1.4 U/g of oil, yielded 99% biodiesel within 48 h.

Molecular bioimprinting of lipases with surfactants and its functional consequences in low water media.

Lipases from Thermomyces lanuginosa (TLL), Candida rugosa (CRL) and Burkholderia cepacia (BCL) were obtained in the 'open lid' form by adding surfactant molecules like n-octyl-ß-d-glucopyranoside (OG), hexadecyl trimethyl ammonium bromide (CTAB), Bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) and triton X-100 for this purpose. The enzymes were 'dried' by precipitating with 4× (v/v) excess of organic solvents. The imprint surfactant molecules were removed by extensive washing with organic solvents. TLL imprinted with 0.05% CTAB showed 11-fold increase in the transesterification activity and was a better preparation to kinetically resolve (±)-1-phenylethanol. Fluorescence emission spectra confirmed that Trp89 of the lid was indeed affected during bioimprinting. With CRL, bioimprinting with OG gave 7-fold increase in the transesterification rates and resulted in reversal of enantioselectivity of CRL and gave R-phenylethyl acetate instead of the S-product as with the unimprinted precipitate. Bioimprinted BCL was also a 7-fold better catalyst for transesterification as well as enantioselectivity.

Structural Modelling, Substrate Binding and Stability Studies of Endopectate Lyase (PL1B) of Family 1 Polysaccharide Lyase from Clostridium thermocellum.

An endo-pectate lyase (PL1B) of family 1 polysaccharide lyase from Clostridium thermocellum was structurally characterized and its stability under chaotropic agent was determined. The putative domain PL1B was identified from the protein sequence ABN53381.1 belonging to superfamily 3 of pectate lyase. Multiple sequence alignment of PL1B with other known pectate lyases revealed the conserved and semi-conserved residues. The secondary structure of PL1B predicted by PsiPred and confirmed by Circular Dichroism showed the presence of 2 α-helices (2.06%), 26 ß-strands (40.54%) and 29 random coils (57.4%). The modelled protein represented right handed parallel ß-helix structure, where three parallel ß-sheets linked by loops coils around to form the ß-helix core. Quality assessment of energy minimized structure by Ramachandran plot displayed 82.8% residues in favoured region. Superposition of PL1B structure with Bsp165-PelA from Bacillus sp. revealed the substrate binding cleft formed by the amino acid residues from the loops and ß-sheet. Molecular dynamic simulation of modelled PL1B structure inferred that it is quite stable and compact. Docking studies identified Asp151, Arg209, Asn234, Arg236, Tyr271 and Ser272 as the key residues of PL1B involved during catalysis. Among them Arg209 is responsible for proton abstraction during ß-elimination. Protein melting studies on PL1B showed that there was 12°C shift of peak from 74 to 86°C in presence of 0.6 mM Ca(2+) ions, showing that they provide stability to the structure. The unfolding of PL1B by GuHCl or Urea by fluorescence study showed that the protein structure is stable and disintegrates at their higher concentrations.

The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.

CtCBM6 of glucuronoxylan-xylanohydrolase (CtXynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ~14 kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that CtCBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of CtCBM6 and that Ca(2+) ions did not affect its structure stability and binding affinity significantly. The CtCBM6 structure was modeled and refined and CD spectrum displayed 44% ß-strands supporting the predicted structure. CtCBM6 displays a jelly roll ß-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting ß4-ß5 and ß8-ß9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of ß-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that CtCBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans.