Production, characterization and techno-economic evaluation of Aspergillus fusant L-asparaginase.

Protoplast fusion is one of the most reliable methods of introducing desirable traits into industrially-promising fungal strains. It harnesses the entire genomic repertoire of fusing microorganisms by routing the natural barrier and genetic incompatibility between them. In the present study, the axenic culture of a thermo-halotolerant strain of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase (L-ASNase) while a xylan-degrading strain of Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Protoplast fusion of the wild strains generated Fusant-06 with improved anti-leukemic and acrylamide reduction potentials. Submerged fed-batch fermentation was preferred to batch and continuous modes on the basis of impressive techno-economics. Fusant-06 L-ASNase was purified by PEG/Na+ citrate aqueous two-phase system (ATPS) to 146.21-fold and global sensitivity analysis report revealed polymer molecular weight and citrate concentration as major determinants of yield and purification factor, respectively. The enzyme was characterized by molecular weight, amino acid profile, activity and stability to chemical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 6.67 × 10-5 M, 1666.67 µmolmin-1 mg-1 protein, 3.88 × 104 min-1 and 5.81 × 108 M-1.min-1 respectively. In-vitro cytotoxicity of HL-60 cell lines by Fusant-06 L-ASNase improved significantly from their respective wild strains. Stability of Fusant-06 L-ASNase over a wide range of pH, temperature and NaCl concentration, coupled with its micromolar Km value, confers commercial and therapeutic value on the product. Free-radical scavenging and acrylamide reduction activities were intermediate and the conferred thermo-halo-stability could be exploited for sustainable clinical and food industry applications.

Interspecific protoplast fusion of atmospheric and room-temperature plasma mutants of Aspergillus generates an L-asparaginase hyper-producing hybrid with techno-economic benefits.

The axenic culture of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase while Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Upon mutagenesis by atmospheric and room-temperature plasma (ARTP), their individual L-asparaginase activities improved 2.3-folds in each of Ile-Thr-Asp-C-180-K and Val-Asp-S-180-E stable mutants. Protoplast fusion of selected stable mutants generated fusant-09 with improved anti-leukemic activity, acrylamide reduction, higher temperature optimum and superior kinetic parameters. Submerged (SmF) and solid-state fermentation (SSF) types were compared; likewise batch, fed-batch and continuous fermentation modes; and fed-batch submerged fermentation was selected on the basis of impressive techno-economics. Fusant L-asparaginase was purified by PEG/Na+ citrate aqueous two-phase system and molecular exclusion chromatography to 69.96 and 146.21-fold, respectively, and characterized by molecular weight, specificity, activity and stability to chemical and physical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 1.667 × 10-3 M, 1666.67 µmol min-1 mg-1 protein, 645.99 s-1 and 3.88 × 105 M-1 s-1 respectively. In-vitro cytotoxicity of HL-60 cell lines by fusant-09 L-asparaginase improved 3.00 and 18.71-folds from mutants Ile-Thr-Asp-C-180-K and Val-Asp-S-180-E, and from 5.73 and 32.55 from respective original strains. Free-radical scavenging and acrylamide reduction improvements were intermediate. Fusant-09 L-asparaginase is strongly recommended for sustainable economic anti-leukemic and food industry applications.

Stochastic modeling and meta-heuristic multivariate optimization of bioprocess conditions for co-valorization of feather and waste frying oil toward prodigiosin production.

Serratia marcescens strain UCCM 00009 produced a mixture of gelatinase and keratinase to facilitate feather degradation but concomitant production of prodigiosin could make waste feather valorization biotechnologically more attractive. This article describes prodigiosin fermentation through co-valorization of waste feather and waste frying peanut oil by S. marcescens UCCM 00009 for anticancer, antioxidant, and esthetic applications. The stochastic conditions for waste feather degradation (WFD), modeled by multi-objective particle swarm-embedded-neural network optimization (ANN-PSO), revealed a gelatinase/keratinase ratio of 1.71 for optimal prodigiosin production and WFD. Luedeking-Piret kinetics revealed a non-exclusive, non-growth-associated prodigiosin yield of 9.66 g/L from the degradation of 88.55% waste feather within 96 h. The polyethylene glycol (PEG) 6000/Na+ citrate aqueous two-phase system-purified serratiopeptidase demonstrated gelatinolytic and keratinolytic activities that were stable for 240 h at 55 °C and pH 9.0. In vitro evaluations revealed that the prodigiosin inhibited methicillin-resistant Staphylococcus aureus at IC50 of 4.95 µg/mL, the plant-pathogen, Sclerotinia sclerotiorum, at IC50 of 2.58 µg/mL, breast carcinoma at IC50 of 0.60 µg/mL and 2,2-diphenyl-1-picryl-hydrazyl hydrate (DPPH) free-radical at IC50 of 96.63 µg/mL). The pigment also demonstrated commendable textile dyeing potential of fiber and cotton fabrics. The technology promises cost-effective prodigiosin development through sustainable waste feather-waste frying oil co-management.

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae: evaluation of production kinetics and enantioselective esterification potential for pharmaceutical applications.

Downstream processing is a significant part of a production process and accounts for 50-90% of the production cost of biotechnological products. Post-fermentation localization of a microbial metabolite contributes significantly to the recovery cost of the product. Enterobacter cloacae produced naturally, acidic lipase with a 0.023:1 extracellular localization ratio. This research aimed to re-direct the localization of lipase to the extracellular milieu to reduce recovery costs using multi-objective response surface optimization (MO-RSM). The approach resulted in a 1:0.32 extracellular: intracellular lipase ratio, with product formation kinetics of Luedeking-Piret function showing a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization. The enzyme was purified by an aqueous two-phase system which extracted 95.22% lipase with 72.36 purity. Characterization of the enzyme showed a molecular weight of 55.7 kDa, kcat of 68.59 s-1, and a Km of 0.63 mmol. Lipase activity occurred optimally at pH 2.5-3.5 and 50 °C, and was stable in most organic solvents tested. The acidic lipase demonstrated pH-dependent enantioselective esterification in resolving (R, S)-ibuprofen (E = 14, pH 4.5) and (R, S)-Naproxen (E = 13, pH 2.5), with an enantioselective preference for (S)-enantiomer in both drugs thus underpinning its potential for pharmaceutical applications.

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae through multi-objective medium optimization: aqueous two-phase purification and activity kinetics.

As physiological impairments that require replacement therapy continue to increase, so also does the need for improved production of acidic lipase from new microbial sources. Enterobacter cloacae strain UCCM 00116 produced a novel acidic lipase in kernel oil-processing waste-basal broth with 0.023:1 extracellular: intracellular localization ratio. This research re-directed enzyme localization to the extracellular milieu to reduce recovery cost using multi-objective response surface optimization of medium parameters. Results revealed a 1:0.32 extracellular:intracellular lipase ratio. Product formation kinetics, modeled by the Luedeking-Piret function, showed a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization through medium optimization. Aqueous two-phase system purification conditions extracted 95.22% lipase with 72.36 purity, a Vmax of 370.37 µmolmin-1, and a Km of 0.63 mmol. Enzyme activity was enhanced by K+ and Ca2+ ions, stable in many organic solvents, except acetone, and had pH and temperature optima at 2.5-3.5 and 50 °C, respectively.

Investing in Late-Life Brain Capital.

Within many societies and cultures around the world, older adults are too often undervalued and underappreciated. This exacerbates many key challenges that older adults may face. It also undermines the many positive aspects of late life that are of tremendous value at both an individual and societal level. We propose a new approach to elevate health and well-being in late life by optimizing late-life Brain Capital. This form of capital prioritizes brain skills and brain health in a brain economy, which the challenges and opportunities of the 21st-century demands. This approach incorporates investing in late-life Brain Capital, developing initiatives focused on building late-life Brain Capital.

Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells.

Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).

Comparative evaluation of antimicrobial efficacy of QMix™ 2 in 1, sodium hypochlorite, and chlorhexidine against Enterococcus faecalis and Candida albicans.

AIM/OBJECTIVE: The aim of this study is to compare the antimicrobial efficacy of QMix™ 2 in 1, sodium hypochlorite (NaOCl), and chlorhexidine (CHX) against Enterococcus faecalis and Candida albicans. MATERIALS AND METHODS: Eighty freshly extracted, single-rooted human mandibular premolar teeth were instrumented and autoclaved. Samples were divided into two groups of 40 teeth each based on the type of microorganism used. Group I was inoculated with E. faecalis and Group II with C. albicans and incubated for 3 days. Each group was subdivided into four subgroups based on the type of irrigant used. Group IA, IIA, 5.25% NaOCl; Group IB, IIB, 2% CHX; Group IC, IIC, QMix™ 2 in 1; and Group ID, IID, 0.9% saline (the control group). Ten microliters of the sample from each canal was taken and was placed on Brain Heart Infusion agar and Sabouraud dextrose agar. The plates were incubated at 37°C for 24 h and colony forming units (CFUs) that were grown were counted. Data was analyzed with analysis of variance (ANOVA) followed by post-hoc Games-Howell test. RESULTS: The greatest antimicrobial effects were observed in samples treated with QMix™ 2 in 1 (P < 0.001). No statistical significant difference was found between 5.25% NaOCl and 2% CHX (P > 0.001) against E. faecalis and C. albicans. CONCLUSION: QMix™ 2 in 1 demonstrated significant antimicrobial efficacy against E. faecalis and C. albicans.

A literature review and case series of accelerating fracture healing in postmenopausal osteoporotic working women.

Majority of fractures do not cause significant long-term morbidity and mortality. A 10% of these fractures result in impaired fracture healing, drastically affecting quality of life in affected patients. Satisfactory healing of these osteoporotic fractures are critically important to functional recovery, morbidity, and quality of life. Some therapies for osteoporosis may affect the processes associated with bone repair. For example, bisphosphonates in experimental models are associated with increased callus size and mineralization, reduced callus remodeling, and improved mechanical strength. Local and systemic bisphosphonate treatment may improve implant fixation. No negative impact on fracture healing has been observed, even after major surgery or when administered immediately after fracture. For the osteoanabolic agent teriparatide, case reports and a randomized trial have produced mixed results, but they are consistent with a positive impact of teriparatide on fracture healing. At this point, therefore, there is no evidence that osteoporosis therapies are detrimental to fracture healing with some promising experimental evidence for positive effects on healing, notably for those agents whose actions are primarily anabolic.

Enhanced genetic modification of adult growth factor mobilized peripheral blood hematopoietic stem and progenitor cells with rapamycin.

Genetic modification of adult human hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors leads to long-term gene expression in the progeny of the HSPCs and has been used to successfully treat several monogenic diseases. In some cases, the gene-modified cells have a selective growth advantage over nonmodified cells and eventually are the dominant engrafted population. However, in disease indications for which the gene-modified cells do not have a selective advantage, optimizing transduction of HSPC is paramount to successful stem cell-based gene therapy. We demonstrate here that transduction of adult CD34+ HSPCs with lentiviral vectors in the presence of rapamycin, a widely used mTORC1 inhibitor, results in an approximately threefold increase in stable gene marking with minimal effects on HSPC growth and differentiation. Using this approach, we have demonstrated that we can enhance the frequency of gene-modified HSPCs that give rise to clonogenic progeny in vitro without excessive increases in the number of vector copies per cell or changes in integration pattern. The genetic marking of HSPCs and expression of transgenes is durable, and transplantation of gene-modified HSPCs into immunodeficient mice results in high levels of gene marking of the lymphoid and myeloid progeny in vivo. The prior safe clinical history of rapamycin in other applications supports the use of this compound to generate gene-modified autologous HSPCs for our HIV gene therapy clinical trials.

Death due to coronary artery insufficiency following blunt trauma to the chest.

There have been numerous reports of sudden cardiac death following blunt trauma to the chest, but there is lack of such references in forensic literature. It is the court of law which makes decision about trauma precipitating natural events. The forensic pathologist is scientifically trained in the medical field and would be in a better position to give clear picture about the victim's general status at the time of death, exact nature and severity of the illness he is suffering from if any. He can also assess the nature and severity of injuries sustained, and thus, his opinion as to the possible role played by the traumatic event in bringing about the death is valuable. This paper will discuss the mechanism of cardiac injury or possible cardiac injury and sudden death of an apparently healthy 36-year-old male following blunt chest trauma sustained during alleged assault by his neighbor.

Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases.

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.

Optimized processing of growth factor mobilized peripheral blood CD34+ products by counterflow centrifugal elutriation.

Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory. Each fraction was analyzed for cell count, viability, and blood cell differential. Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions. With modification of the basic protocol, we were able to collect all of the CD34+ cells in a single fraction. The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34, washed using the Elutra device, and transferred directly to a magnetic bead selection device for further purification. CD34+ cell purities from the column were extremely high (98.7 ± 0.9%), and yields were typical for the device (55.7 ± 12.3%). The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.

Health problems among menopausal women in Udupi district (Karnataka).

Menopause among women, occurring in middle age, brings in its wake, a set of health problems that needs to be handled distinctly by the care givers. A study undertaken to determine the magnitude of health problems in Udupi district of Karnataka included 100 menopausal women in the age group 45-55 years, 50 each from urban and rural pockets. Using demographic proforma, modified socio-economic scale and structured interview schedule as tools, it was concluded that menopausal health problems were more common in women in rural areas than in their urban counterparts: they were also less articulate and less aware about managing or preventing menopausal health problems.

RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma.

AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell-based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood-derived (CD34(+)) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to approximately 1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.