Venkata Raveendra Pothineni

From solvent-free microspheres to bioactive gradient scaffolds

A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore size gradient for tissue engineering applications. Poly(D,L-Lactide) microspheres were fabricated through an emulsification method where TiO2 nanoparticles were employed both as particulate emulsifier in the preparation procedure and as surface modification agent to improve bioactivity of the scaffolds. A fine-tunable pore size gradient was achieved with a pore volume of 30±2.6%. SEM, EDX, XRD and FTIR analyses all confirmed the formation of bone-like apatite at the 14th day of immersion in Simulated Body Fluid (SBF) implying the ability of our scaffolds to bond to living bone tissue. In vitro examination of the scaffolds showed progressive activity of the osteoblasts on the scaffold with evidence of increase in its mineral content. The bioactive scaffold developed in this study has the potential to be used as a suitable biomaterial for bone tissue engineering and hard tissue regeneration.

Prolonged survival of transplanted stem cells after ischaemic injury via the slow release of pro-survival peptides from a collagen matrix

Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen–dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.The slow release of pro-survival peptide analogues crosslinked to an injectable collagen–dendrimer biomaterial significantly prolongs the engraftment and survival of transplanted stem cells in mouse models of ischaemic injury.

Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening

Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%–20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited >90% of B. burgdorferi growth at a concentration of <25 µM. These 150 unique compounds comprise many safe antibiotics, chemical compounds, and also small molecules from plant sources. Of the 150 unique compounds, 101 compounds are FDA approved. We selected the top 20 FDA-approved molecules based on safety and potency and studied their minimum inhibitory concentration and minimum bactericidal concentration. The promising safe FDA-approved candidates that show low minimum inhibitory concentration and minimum bactericidal concentration values can be chosen as lead molecules for further advanced studies.

Borreliacidal activity of Borrelia metal transporter A (BmtA) binding small molecules by manganese transport inhibition.

Borrelia burgdorferi, the causative agent of Lyme disease, utilizes manganese (Mn) for its various metabolic needs. We hypothesized that blocking Mn transporter could be a possible approach to inhibit metabolic activity of this pathogen and eliminate the infection. We used a combination of in silico protein structure prediction together with molecular docking to target the Borrelia metal transporter A (BmtA), a single known Mn transporter in Borrelia and screened libraries of FDA approved compounds that could potentially bind to the predicted BmtA structure with high affinity. Tricyclic antihistamines such as loratadine, desloratadine, and 3-hydroxydesloratadine as well as yohimbine and tadalafil demonstrated a tight binding to the in silico folded BmtA transporter. We, then, tested borreliacidal activity and dose response of the shortlisted compounds from this screen using a series of in vitro assays. Amongst the probed compounds, desloratadine exhibited potent borreliacidal activity in vitro at and above 78 μg/mL (250 μM). Borrelia treated with lethal doses of desloratadine exhibited a significant loss of intracellular Mn specifically and a severe structural damage to the bacterial cell wall. Our results support the possibility of developing a novel, targeted therapy to treat Lyme disease by targeting specific metabolic needs of Borrelia.

Microhemorrhage-associated tissue iron enhances the risk for Aspergillus fumigatus invasion in a mouse model of airway transplantation

Increased ferric iron in allografts triggers a switch in the growth of the mold Aspergillus fumigatus from a colonizing to an invasive phenotype. Irons in the fire Although transplantation is a lifesaving therapy, patients receiving new organs are at serious risk for invasive, potentially fatal infections. Aspergillus fumigatus is a particularly common and troublesome fungal pathogen, but its ability to invade transplant tissues is poorly understood. To evaluate this property, Hsu and colleagues infected transplants in mice. Bleeding, caused by damage to small vessels in grafted airways, led to increased tissue iron, a known growth factor for Aspergillus. Increased tissue iron is a newly identified risk factor for transplant damage by microorganisms. Therapies in development that block iron and protect blood vessels may extend the life of organ recipients. Invasive pulmonary disease due to the mold Aspergillus fumigatus can be life-threatening in lung transplant recipients, but the risk factors remain poorly understood. To study this process, we used a tracheal allograft mouse model that recapitulates large airway changes observed in patients undergoing lung transplantation. We report that microhemorrhage-related iron content may be a major determinant of A. fumigatus invasion and, consequently, its virulence. Invasive growth was increased during progressive alloimmune-mediated graft rejection associated with high concentrations of ferric iron in the graft. The role of iron in A. fumigatus invasive growth was further confirmed by showing that this invasive phenotype was increased in tracheal transplants from donor mice lacking the hemochromatosis gene (Hfe−/−). The invasive phenotype was also increased in mouse syngrafts treated with topical iron solution and in allograft recipients receiving deferoxamine, a chelator that increases iron bioavailability to the mold. The invasive growth of the iron-intolerant A. fumigatus double-knockout mutant (ΔsreA/ΔcccA) was lower than that of the wild-type mold. Alloimmune-mediated microvascular damage and iron overload did not appear to impair the host’s immune response. In human lung transplant recipients, positive staining for iron in lung transplant tissue was more commonly seen in endobronchial biopsy sections from transplanted airways than in biopsies from the patients’ own airways. Collectively, these data identify iron as a major determinant of A. fumigatus invasive growth and a potential target to treat or prevent A. fumigatus infections in lung transplant patients.

Omics Approaches in Industrial Biotechnology and Bioprocess Engineering

Abstract The constant uncontrolled rise in the population calls for the efforts to ensure the timely availability of biopharmaceuticals to address the prophylactic and therapeutic needs of the masses. This, hence, calls for the constant upgradation and optimization of the industrial bioprocesses. Improving the output of bioprocesses to make them more cost effective and time efficient requires the incorporation and use of newer techniques. Most industrial biotechnological products involve the involvement of at least one biological organism. Therefore in order to enhance the output of these processes, an intelligent optimization of the bioprocesses has to be carried out. The advent of omics has been a tantamount to the progress in all the fields of biotechnology. A number of tools have been employed by the biotechnologists, systems biologists, and bioprocess engineers for optimizing and developing efficient industrial process for biological applications. The efficient production of quality biological products is based on the selection, optimization, and engineering of appropriate biological mechanisms. Recent advances in the field of genomics, transcriptomics, proteomics, and metabolomics have led to an overall increase in the industrial output and, hence, an improvement in the biomedical outcomes. This chapter addresses the history of the inculcation of omics technologies in the field of industrial biotechnology followed by the tools developed for improving the industrial output. The challenges faced in this sector have also been addressed.

Screening of NCI-DTP Library to Identify New Drug Candidates for Borrelia burgdorferi

Lyme disease is the most rapidly growing tick borne zoonotic disease of the Northern Hemisphere and is among the 10 most commonly reported nationally notifiable diseases in the United States.1 Clinical presentations include erythema migrans, fever, chills, muscle and joint pain.2,3 Though these symptoms tend to fade away even without therapeutic intervention, a significant number of untreated patients develop arthritis and persistent myalgia following exposure to Borrelia burgdorferi.4 Furthermore, 10–20% of patients treated for Lyme disease develop symptoms considered typical, or even exaggerated, including muscle, joint pain and generalized fatigue5,6. This condition is referred as post-treatment lyme disease syndrome (PTLDS). Though existence of PTLDS is debatable, some researchers consider the presence of persister forms of B. burgdorferi and/or the continuous presence of antigenic debris to be the underlying cause of PTLDS.7–10 Like other pathogens, B. burgdorferi protects itself from the immune system and from drug treatment.11,12 B. burgdorferi evades immune response by antigenic variation of its surface proteins13–15. In a recent study, researchers identified B. burgdorferi persisters in in vitro cultures.12 They found the killing of B. burgdorferi by antibiotics is biphasic, with a small subpopulation of surviving persisters.12 The surviving antibiotic tolerant cells are not resistant mutants upon regrowth, the population bifurcates into new antibiotic-susceptible and new persister subpopulations.12 Currently prescribed drugs for treating Lyme disease, including amoxicillin, ceftriaxone and doxycycline were unable to completely eliminate the B. burgdorferi.12,16–18 So, efforts to identify new, potent drug candidates for Lyme disease are on the rise. Many researchers are performing high-throughput screening of drugs against B. burgdorferi persisters to identify molecules that can eliminate complete Borrelial infection.7,17–20 Screening of chemical compound libraries serves to test a large number of structurally and functionally diverse molecules against pathogenic agents. Among the many chemical libraries currently available, the Developmental Therapeutics Program of the National Cancer Institute, National Institute of Health, provides a unique yet diverse array of chemical compounds for screening purpose. Four sets of compounds within the National Cancer InstituteDevelopmental Therapeutics Program (NCI-DTP) library (http://dtp. nci.nih.gov/) tend to represent a wide variety of structural and functional diversity. This study aimed to identify new, effective drugs for Lyme disease. We used a well-established, highly efficient, BacTiter-Glo assay (Promega Corporation, Fitchburg, WI, USA) which can detect as few as 7 × 103 Borrelial cells in BSK-II medium.21,22 The NCI-DTP compound library of four diverse sets containing 3084 chemical compounds was screened using this assay. We identified 101 unique compounds which inhibited Borrelia growth by more than 85% at or below a concentration of 25 μM. From these 101 compounds we selected 12 molecules and studied their MIC and MBC. The lead compounds identified in the current study can be further evaluated for their therapeutic potential in pre-clinical and clinical studies. Moreover, the outcomes of the study could provide a deeper insight into treatment strategies for Lyme disease. We have developed a one-step, straightforward, highly sensitive BacTiter-Glo (Promega Corporation) Assay to screen drugs in highthroughput format. We optimized a BacTiter-Glo (Promega Corporation) Assay in high-throughput screening format as reported in our previous papers.21,22 The BacTiter-Glo Assay (Promega Corporation) assesses bacterial viability by measuring ATP in the sample. This sensitive assay can reliably detect as few as 10 Borrelia cells in phosphate buffered saline or 7 × 103 Borrelia cells in BSK-II medium21,22. By using this BacTiter-Glo (Promega Corporation) Assay we have screened NCI-DTP library containing 3084 chemical compounds.22 The NCI-DTP compound library we have screened contains a total of 3084 chemical compounds from four highly divergent sets viz, Structural diversity set (1974 compounds),

Application of Bioinformatics and System Biology in Medicinal Plant Studies

Bioinformatics and systems biology has adapted an essential role in the study of medicinal plants today. With an ever-increasing genomic and proteomic information, it is necessary to interpret the data adequately. Bioinformatics has led to the discovery of new genetic players and helped in the characterization of many of the new genes and proteins. Moreover, its tools have helped in deciphering important relationships between the various molecular players. However, as the data increases exponentially, a parallel growth of the bioinformatics tools and methods is inevitable. There is a constant need to develop new means to carry out data management, analysis, visualization, and prediction. In this chapter, the key applications of bioinformatics in understanding medicinal plant biology have been discussed with a special emphasis on the incorporation of bioinformatics tools and methods. All these aspects have been covered in the light of omics technologies including genomics, transcriptomics, proteomics, and metabolomics. Toward the end of the chapter, the current and future trends in the field of bioinformatics pertaining to medicinal plants have been discussed.