Limb reconstruction by free-tissue transfer combined with the Ilizarov method.

In four complex cases of extremity reconstruction, we have been able to overcome the problems of combined bone and soft-tissue loss and length discrepancy by a combination of free-tissue transfer and the Ilizarov method of slow distraction. It is our observation that gradual distraction of a free tissue is a safe and viable procedure; the free tissue tolerates the pins of the circular external fixator well, and there is an equal degree of distraction and regeneration of the transferred free tissue and the native recipient tissue without evidence of wound dehiscence. Corticotomy through free tissue and in close proximity to vascularized bone is safe, with the subsequent bone regeneration not unlike that of normal bone. Manipulation by slow distraction does not appear to compromise the vasculature of the recipient bed for later microsurgical procedures or endanger the axial flow pattern of the transferred free tissue.     

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.     

Modifications of Golgi complex in chondrocytes from osteoarthrotic (OA) rat cartilage.

The status of the Golgi complex in normal vs osteoarthrotic (OA) cartilage has not yet been studied. A monoclonal antibody, MAb 58-K-9, allowed scoring of Golgi labeling intensity. In addition, ultrastructural assessment enabled us to focus on the distribution and relation between the endoplasmic reticulum (ER) and Golgi membranes. The study was performed in both normal and partially menisectomized OA-induced rat cartilage 20 and 45 days after surgery. Comparing Golgi immunolabeling intensities (mean +/- SEM) revealed a highly significant difference between normal (9.98 +/- 1.25), 20-day (2.49 +/- 0.34), and 45-day (0.82 +/- 0.22) cartilage. Moreover, chondrocytes from normal cartilage displayed 71.18% of labeling intensity in contrast to OA cartilage, in which chondrocyte labeling intensities were 24.95% (20 days) and 8.11% (45 days). OA chondrocytes appeared to display an overall reduction in Golgi labeling intensity, suggesting disruption of this organelle as the OA damage progressed. Interestingly, many 20-day OA-induced chondrocytes exhibited bubble-like Golgi immunolabeling compartmentalizing the cytoplasm, concomitant with putative apoptotic nuclear changes. At the same time, OA chondrocytes with a typical ultrastructural apoptotic pattern revealed a prominent ER gathered together with Golgi vesicles and saccules, also appearing to compartmentalize chondrocyte cytoplasm. We speculate about the role of Golgi modifications and apoptosis in OA pathogenesis.     

An endophytic Neurospora sp. from Nothapodytes foetida producing camptothecin

The medicinal plant, Nothapodytes foetida contains a number of important alkaloids like camptothecin (an anticancer drug molecule) but its concentration is less to meet the existing demand of this important molecule, so in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as potential source of anticancer drug lead compound i.e. camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of precursor anticancer drug molecule in future at large scale.     

Minerals solubilizing and mobilizing microbiomes: A sustainable approach for managing minerals’ deficiency in agricultural soil

Agriculture faces challenges to fulfil the rising food demand due to shortage of arable land and various environmental stressors. Traditional farming technologies help in fulfilling food demand but they are harmful to humans and environmental sustainability. The food production along with agro-environmental sustainability could be achieved by encouraging farmers to use agro-environmental sustainable products such as biofertilizers and biopesticides consisting of live microbes or plant extract instead of chemical-based inputs. The eco-friendly formulations play a significant role in plant growth promotion, crop yield and repairing degraded soil texture and fertility sustainably. Mineral solubilizing microbes that provide vital nutrients like phosphorus, potassium, zinc and selenium are essential for plant growth and development and could be developed as biofertilizers. These microbes could be plant associated (rhizospheric, endophytic and phyllospheric) or inhabit the bulk soil and diverse extreme habitats. Mineral solubilizing microbes from soil, extreme environments, surface and internal parts of the plant belong to diverse phyla such as Ascomycota, Actinobacteria, Basidiomycota, Bacteroidetes, Chlorobi, Cyanobacteria, Chlorophyta, Euryarchaeota, Firmicutes, Gemmatimonadetes, Mucoromycota, Proteobacteria and Tenericutes. Mineral solubilizing microbes (MSMs) directly or indirectly stimulate plant growth and development either by releasing plant growth regulators; solubilizing phosphorus, potassium, zinc, selenium and silicon; biological nitrogen fixation and production of siderophores, ammonia, hydrogen cyanide, hydrolytic enzymes and bioactive compound/secondary metabolites. Biofertilizer developed using mineral solubilizing microbes is an eco-friendly solution to the sustainable food production system in many countries worldwide. The present review deals with the biodiversity of mineral solubilizing microbes, and potential roles in crop improvement and soil well-being for agricultural sustainability.     

Epigenetics DNA methylation in the core ataxin-2 gene promoter: novel physiological and pathological implications

Pathogenic CAG (cytosine-adenine-guanine) expansions beyond certain thresholds in the ataxin-2 (ATXN2) gene cause spinocerebellar ataxia type 2 (SCA2) and were shown to contribute to Parkinson disease, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Regulation of ATXN2 gene expression and the function of the protein product are not known. SCA2 exhibits an inverse correlation between the size of the CAG repeat and the age at disease onset. However, a wide range of age at onset are typically observed, with CAG repeat number alone explaining only partly this variability. In this study, we explored the hypothesis that ATXN2 levels could be controlled by DNA methylation and that the derangement of this control may lead to escalation of disease severity and influencing the age at onset. We found that CpG methylation in human ATXN2 gene promoter is associated with pathogenic CAG expansions in SCA2 patients. Different levels of methylation in a SCA2 pedigree without an intergenerational CAG repeat instability caused the disease anticipation in a SCA2 family. DNA methylation also influenced the disease onset in SCA2 homozygotes and SCA3 patients. In conclusion, our study points to a novel regulatory mechanism of ATXN2 expression involving an epigenetic event resulting in differential disease course in SCA2 patients.     

Molecular identification and phylogenetic analyses of multiple nucleopolyhedrovirus isolated from <Emphasis Type="Italic">Lymantria obfuscata</Emphasis> (Lepidoptera: Lymantriidae) in India

The Lymantria obfuscata Walker (Lyob) multiple (M) nucleopolyhedrovirus (NPV) (LyobMNPV) has been isolated and successfully applied for the management of the Indian gypsy moth, L. obfuscata in Jammu and Kashmir (J&K), India. The present work aimed to investigate the variability of LyobMNPV isolates from six localities of J&K through molecular [amplification of the polyhedrin (polh), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes] and biological (bioassays) characterization. To identify the position of LyobMNPV in the phylogenetic tree of baculoviruses, partial sequences of the polh, lef-8 and lef-9 genes were determined by using the DNA sequences within their coding regions by optimizing the polymerase chain reaction with degenerate primers. The sequence alignment revealed that LyobMNPV isolates exhibited seven, five and eleven single nucleotide polymorphic sites in the case of polh, lef-8 and lef-9, respectively. The phylogenetic analyses supported placing LyobMNPV with the Lymantria dispar L. MNPV (LdMNPV) isolates from different countries, and showed that it was more closely related to LdMNPV than to Lymantria xylina Swinhoe NPV and Lymantria monacha L. NPV. The contaminated diet plug bioassays using 2nd instar larvae indicated that the median lethal dose (LD₅₀) and median survival time (ST₅₀) of different isolates of LyobMNPV against L. obfuscata were lower than those of LdMNPV against L. dispar. LyobMNPV was more closely related to LdMNPV but its LD₅₀ and ST₅₀ were lower than those of LdMNPV. The study provides novel information on the position of LyobMNPV in the phylogenetic tree of baculoviruses and about biological and genetic variation of Lymantria species’ NPV isolates from different parts of the world.     

<Emphasis Type="Italic">In vitro</Emphasis> cytotoxicity of an endophytic fungus isolated from<Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

An attempt has been made to assessin vitro cytotoxicity of an endophytic fungus fromNothapodytes foetida. Various human cancer cell lines (liver HEP-2, lung A-549, ovary OVAR-5, prostate PC-3, cervix Hela, colon HCT-15, oral cell line KB, CNS SNB-78, were used.In vitro cytotoxicity of camptothecin (CPT) isolated from the fungus was done where OVAR-5 cell line showed maximum inhibition and HEP-2 cell line was least sensitive with this compound.In vitro cytotoxicity of fractions/extracts from endophyte was carried out where ethyl acetate fraction showed sufficient growth inhibition against all the cell lines.