Investigating alterations in the cellular envelope of Staphylococcus aureus in simulated microgravity using a random positioning machine.

Continuous rotation of liquid bacterial culture in random positioning machine (RPM) causes formation of a colloidal bacterial culture in the culture tube, due to lack of sedimentation and convection. Interestingly, similar colloidal bacterial cultures can also be seen in suspended bacterial cultures in a spaceflight environment. Thus, as a consequence of no sedimentation, an alteration in the microenvironment of each bacterial cell in simulated microgravity is introduced, compared to the bacterial culture grown in normal gravity wherein they sediment slowly at the bottom of the culture tube. Apparently, a bacterial cell can sense changes in its environment through various receptors and sensors present at its surface, thus it can be speculated that this change in its microenvironment might induce changes in its cell wall and cell surface properties. In our study, changes in growth kinetics, cell wall constitution using FTIR (Fourier Transform Infrared Spectroscopy), cell surface hydrophobicity, autoaggregation ability and antibiotic susceptibility of Staphylococcus aureus NCIM 2079 strain, in simulated microgravity (using RPM) was studied in detail. Noteworthy alterations in its growth kinetics, cell wall constitution, cell surface hydrophobicity, autoaggregation ability and antibiotic susceptibility especially to Erythromycin and Clindamycin were observed. Our data suggests that microgravity may cause alterations in the cellular envelope of planktonic S.aureus cultures.

Response of extreme haloarchaeon Haloarcula argentinensis RR10 to simulated microgravity in clinorotation.

Gravity is the fundamental force that may have operated during the evolution of life on Earth. It is thus important to understand as to what the effects of gravity are on cellular life. The studies related to effect of microgravity on cells may provide greater insights in understanding of how the physical force of gravity shaped life on Earth. The present study focuses on a unique group of organisms called the Haloarchaea, which are known for their extreme resistance to survive in stress-induced environments. The aim of the present investigation was to study the effect of simulated microgravity on physiological response of extremely halophilic archaeon, Haloarcula argentinensis RR10, under slow clinorotation. The growth kinetics of the archaeon in microgravity was studied using the Baryani model and the viable and apoptotic cells were assessed using propidium iodide fluorescent microscopic studies. The physiological mechanism of adaptation was activation of 'salt-in' strategy by intracellular sequestration of sodium ions as detected by EDAX. The organism upregulated the production of ribosomal proteins in simulated microgravity as evidenced by Matrix-assisted laser desorption ionization Time of flight-Mass Spectrophotometry. Simulated microgravity altered the antibiotic susceptibility of the haloarchaeon and it developed resistance to Augmentin, Norfloxacin, Tobramycin and Cefoperazone, rendering it a multidrug resistant strain. The presence of antibiotic efflux pump was detected in the haloarchaeon and it also enhanced production of protective carotenoid pigment in simulated microgravity. The present study is presumably the first report of physiological response of H. argentinensis RR10 in microgravity simulated under slow clinorotation.

Studies on efficacy of a novel 177Lu-labeled porphyrin derivative in regression of tumors in mouse model.

OBJECTIVE: The aim of the present study was to develop a (177)Lu-labeled porphyrin derivative having favorable characteristics for use in targeted radiotherapy of cancer and to evaluate its biological behavior in mouse tumor models with respect to its effectiveness in tumor regression. Owing to the inherent affinity of porphyrins to accumulate in the tumors, suitably modified porphyrin derivative was chosen as the vehicle for the targeted delivery of the radionuclide. (177)Lu was preferred as the radionuclide of choice due to its suitable nuclear decay characteristics [E(ß(max)) = 497 keV, Eγ = 208 keV (11%), 113 keV (6.4%)], comparatively longer half-life (6.73 d) and ease of production in adequate quantity and sufficiently high specific activity using medium flux research reactors. METHODS: A novel porphyrin analogue, 5,10,15,20-tetrakis[4-carboxymethyleneoxyphenyl]porphyrin was synthesized inhouse and coupled with a macrocyclic bi-functional chelating agent, namely p-amino-benzyl-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid. The porphyrin-BFCA conjugate was labeled with (177)Lu and the biological behavior of the radiolabeled conjugate was studied by biodistribution and imaging in Swiss mice bearing either fibrosarcoma or thymic lymphoma tumors. Effectiveness of the agent in controlling the growth of tumor volumes was also studied by administering various doses of the radiolabeled preparation in the mouse tumor models. RESULTS: (177)Lu-labeled porphyrin-BFCA conjugate was prepared with high radiochemical purity ( > 99%) and adequate invitro stability. Biodistribution and imaging studies revealed good uptake and retention of the agent in the tumors with encouraging tumor to blood and tumor to muscle ratios at various post-administration time points. Tumor regression studies showed that the administration of the agent increased the average tumor doubling time and decreased the average specific growth rate substantially in both the types of tumors. However, thymic lymphoma was found to be more sensitive to the radiolabeled conjugate compared to fibrosarcoma. CONCLUSION: Preliminary biological evaluation and tumor regression studies carried out in two different tumor models in Swiss mice exhibited the promising nature of (177)Lu-labeled porphyrin-BFCA conjugate as an agent for targeted tumor therapy. However, further detailed investigations are warranted to evaluate the true potential of the developed agent.

Biologic evaluation of a novel 188Re-labeled porphyrin in mice tumor model.

The aim of this study was to develop a (188)Re-labeled porphyrin-based tumor-specific agent and to evaluate its biologic behavior, including tumor-regressing effectiveness, in mouse tumor models for possible use in achieving targeted cancer radiotherapy. (188)Re was obtained from an alumina-column-based (188)W-(188)Re generator constructed in-house. The compound, 5,10,15,20-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin, was synthesized and labeled with (188)ReO(4)(-). (188)Re-labeled porphyrin complex was produced with a radiochemical purity of approximately 98% with reasonably good in vitro stability (>24 hours at 4 degrees C). Swiss mice bearing thymic lymphoma and fibrosarcoma were used as tumor models. The biodistribution studies revealed satisfactory tumor retention (2.07% +/- 0.80% injected activity per g) with insignificant activities in blood (0.53%), liver (0.26%) and kidney (0.04%) at 24 hours. The radiolabeled conjugate treatment increased the average tumor-doubling time and decreased the average specific growth rate substantially in thymic lymphoma, compared to fibrosarcoma tumor. (188)Re-labeled 5,10,15,20-tetrakis[3,4 bis(carboxymethyleneoxy)phenyl] porphyrin has specific affinity toward the fibrosarcoma and thymic lymphoma tumors in mice. Thymic lymphoma was found to be more sensitive to the radionuclide complex, compared to fibrosarcoma. The (188)Re-labeled porphyrin complex showed promising results and warrants further investigations.

FBX aqueous chemical dosimeter for measurement of virtual wedge profiles.

We investigated the ferrous sulfate-benzoic acid-xylenol orange (FBX) aqueous chemical dosimeter for measurement of virtual (dynamic) wedge profiles on a linear accelerator. The layout for irradiation of the FBX-filled tubes mimicked a conventional linear detector array geometry. A comparison of the resulting measurements with film-measured profiles showed that, in the main beam region, the difference between the FBX system and the film system was within +/-2% and that, in the penumbra region, the difference varied from +/-1 mm to +/-2.5 mm in terms of positional equivalence, depending on the size of the dosimeter tubes. We thus believe that the energy-independent FBX dosimetry system can measure virtual wedge profiles with reasonable accuracy at reasonable cost. However, efficiency improvement is required before this dosimetry system can be accepted into routine practice.

ISHAN: sequence homology analysis package.

Sequence based homology studies play an important role in evolutionary tracing and classification of proteins. Various methods are available to analyze biological sequence information. However, with the advent of proteomics era, there is a growing demand for analysis of huge amount of biological sequence information, and it has become necessary to have programs that would provide speedy analysis. ISHAN has been developed as a homology analysis package, built on various sequence analysis tools viz FASTA, ALIGN, CLUSTALW, PHYLIP and CODONW (for DNA sequences). This JAVA application offers the user choice of analysis tools. For testing, ISHAN was applied to perform phylogenetic analysis for sets of Caspase 3 DNA sequences and NF-kappaB p105 amino acid sequences. By integrating several tools it has made analysis much faster and reduced manual intervention.

Electroporation enhances radiation and doxorubicin-induced toxicity in solid tumor in vivo.

Treatment of cancer patients is subject to limitations in radiotherapy and chemotherapy. This necessitates development of new protocols, and the present work reports on the effects of a combination of local electroporation with ionizing radiation and/or anticancer drug doxorubicin hydrochloride (DOX) on subcutaneous solid tumor murine fibrosarcoma. Localized treatment of fibrosarcoma tumor, grown in right hind leg of Swiss mice, has been carried out using DOX (0.6 mg/kg body weight), radiation (Co 60 gamma-rays, dose rate 0.37 Gy/min) and electroporation (1 kV/cm, 200 micros, 8 pulses per burst, 10 bursts) individually or in combinations. Measurements of the tumor growth kinetics after treatment with combinations have revealed significant growth delay. The treatment groups, (i) radiation and electroporation, (ii) DOX and electroporation, and (iii) radiation, DOX and electroporation, have yielded tumor growth delays (TGDs) of 1.22, 1.5, and 1.73 days, respectively, compared to control with the tumor volumes being 53%, 57%, and 49% that of control on the final day of observation. These results suggest that the antitumor effects of a moderate dose of gamma radiation and low concentration of DOX can be significantly enhanced by combination with electroporation.

Enhancement of radiation cytotoxicity in murine cancer cells by electroporation: in vitro and in vivo studies.

Increasing evidence has accumulated in recent years to suggest that the cell membrane forms the vital common target for action by ionizing radiation and electroporation. The present work describes the use of electric pulses for enhancement of radiation-induced cytotoxicity of cancer cells both in vitro and in vivo. In vitro: low dose rate (0.37 Gy/min) Co60 gamma-rays (2 Gy) in combination with electric pulses of square wave (2 kV/cm, 200 micros duration, 8 pulses/burst, 10 times) significantly enhanced the cytotoxicity in Ehrlich ascites carcinoma cells (EAC), probably through enhanced production of intracellular reactive oxygen species (ROS). The intracellular generation of ROS and changes in oxidative damage-mediated membrane fluidity were determined by fluorescence using DCH-FDA and DPH, respectively, as probes. Both radiation and electroporation, separately, have been observed to produce ROS in a dose-dependent fashion. We show that the combined treatment of cells with radiation and electroporation significantly increased intracellular ROS and changed membrane fluidity of EAC cells as compared to the effects by each individual treatment. In vivo studies have been carried out with murine fibrosarcoma as a model system. The localized treatment of a fibrosarcoma tumor, grown in the right hind leg of Swiss mice, had been carried out using radiation (Co60 gamma-rays, 2 Gy, dose rate: 0.37 Gy/min) and electric pulses (1 kV/cm, 200 micros, 8 pulses/burst, 10 times). Studies on tumor growth kinetics have shown a significant growth delay (by 50% to that of control) 7 days after treatment of tumor with radiation and electroporation. The results suggest that radiocytotoxicity of tumor cells in vitro as well as in vivo were enhanced significantly by electric pulses, which may offer a potentially improved treatment of cancer.