De novo transcriptome assembly and global analysis of differential gene expression of aphid tolerant wild mustard Rorippa indica (L.) Hiern infested by mustard aphid Lipaphis Erysimi (L.) Kaltenbach.

Rapeseed-mustard, the oleiferous Brassica species are important oilseed crops cultivated all over the globe. Mustard aphid Lipaphis erysimi (L.) Kaltenbach is a major threat to the cultivation of rapeseed-mustard. Wild mustard Rorippa indica (L.) Hiern shows tolerance to mustard aphids as a nonhost and hence is an important source for the bioprospecting of potential resistance genes and defense measures to manage mustard aphids sustainably. We performed mRNA sequencing of the R. indica plant uninfested and infested by the mustard aphids, harvested at 24 hours post-infestation. Following quality control, the high-quality reads were subjected to de novo assembly of the transcriptome. As there is no genomic information available for this potential wild plant, the raw reads will be useful for further bioinformatics analysis and the sequence information of the assembled transcripts will be helpful to design primers for the characterization of specific gene sequences. In this study, we also used the generated resource to comprehensively analyse the global profile of differential gene expression in R. indica in response to infestation by mustard aphids. The functional enrichment analysis of the differentially expressed genes reveals a significant immune response and suggests the possibility of chitin-induced defense signaling.

Enhancement of ABA Sensitivity Through Conditional Expression of the ARF10 Gene in Brassica juncea Reveals Fertile Plants with Tolerance Against Alternaria brassicicola.

Concomitant increase of auxin-responsive factors ARF16 and ARF17, along with enhanced expression of ARF10 in resistant Sinapis alba compared with that in susceptible Brassica juncea upon challenge with Alternaria brassicicola, revealed that abscisic acid (ABA)-auxin crosstalk is a critical factor for resistance response. Here, we induced the ABA response through conditional expression of ARF10 in B. juncea using the A. brassicicola-inducible GH3.3 promoter. Induced ABA sensitivity caused by conditional expression of ARF10 in transgenic B. juncea resulted in tolerance against A. brassicicola and led to enhanced expression of several ABA-responsive genes without affecting the auxin biosynthetic gene expression. Compared with ABI3 and ABI4, ABI5 showed maximum upregulation in the most tolerant transgenic lines upon pathogen challenge. Moreover, elevated expression of ARF10 by different means revealed a direct correlation between ARF10 expression and the induction of ABI5 protein in B. juncea. Through in vitro DNA-protein experiments and chromosome immunoprecipitation using the ARF10 antibody, we demonstrated that ARF10 interacts with the auxin-responsive elements of the ABI5 promoter. This suggests that ARF10 may function as a modulator of ABI5 to induce ABA sensitivity and mediate the resistance response against A. brassicicola.

Chickpea WRKY70 Regulates the Expression of a Homeodomain-Leucine Zipper (HD-Zip) I Transcription Factor CaHDZ12, which Confers Abiotic Stress Tolerance in Transgenic Tobacco and Chickpea.

Drought and salinity are the two major environmental constraints that severely affect global agricultural productivity. Plant-specific HD-Zip transcription factors are involved in plant growth, development and stress responses. In the present study, we explored the functional characteristics and regulation of a novel HD-Zip (I) gene from chickpea, CaHDZ12, in response to water-deficit and salt-stress conditions. Transgenic tobacco lines over-expressing CaHDZ12 exhibited improved tolerance to osmotic stresses and increased sensitivity to abscisic acid (ABA). Physiological compatibility of transgenic lines was found to be more robust compared to the wild-type plants under drought and salinity stress. Additionally, expression of several stress-responsive genes was significantly induced in CaHDZ12 transgenic plants. On the other hand, silencing of CaHDZ12 in chickpea resulted in increased sensitivity to salt and drought stresses. Analysis of different promoter deletion mutants identified CaWRKY70 transcription factor as a transcriptional regulator of CaHDZ12 expression. In vivo and in vitro interaction studies detected an association between CaWRKY70 and CaHDZ12 promoter during stress responses. Epigenetic modifications underlying histone acetylation at the CaHDZ12 promoter region play a significant role in stress-induced activation of this gene. Collectively, our study describes a crucial and unique mechanistic link between two distinct transcription factors in regulating plant adaptive stress response.

Analysis of root proteome unravels differential molecular responses during compatible and incompatible interaction between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp. ciceri Race1 (Foc1).

BACKGROUND: Vascular wilt caused by Fusarium oxysporum f. sp. ciceri Race 1 (Foc1) is a serious disease of chickpea (Cicer arietinum L.) accounting for approximately 10-15% annual crop loss. The fungus invades the plant via roots, colonizes the xylem vessels and prevents the upward translocation of water and nutrients, finally resulting in wilting of the entire plant. Although comparative transcriptomic profiling have highlighted some important signaling molecules, but proteomic studies involving chickpea-Foc1 are limited. The present study focuses on comparative root proteomics of susceptible (JG62) and resistant (WR315) chickpea genotypes infected with Foc1, to understand the mechanistic basis of susceptibility and/or resistance. RESULTS: The differential and unique proteins of both genotypes were identified at 48 h, 72 h, and 96 h post Foc1 inoculation. 2D PAGE analyses followed by MALDI-TOF MS and MS/MS identified 100 differentially (>1.5 fold<, p<0.05) or uniquely expressed proteins. These proteins were further categorized into 10 functional classes and grouped into GO (gene ontology) categories. Network analyses of identified proteins revealed intra and inter relationship of these proteins with their neighbors as well as their association with different defense signaling pathways. qRT-PCR analyses were performed to correlate the mRNA and protein levels of some proteins of representative classes. CONCLUSIONS: The differential and unique proteins identified indicate their involvement in early defense signaling of the host. Comparative analyses of expression profiles of obtained proteins suggest that albeit some common components participate in early defense signaling in both susceptible and resistant genotypes, but their roles and regulation differ in case of compatible and/or incompatible interactions. Thus, functional characterization of identified PR proteins (PR1, BGL2, TLP), Trypsin protease inhibitor, ABA responsive protein, cysteine protease, protein disulphide isomerase, ripening related protein and albumins are expected to serve as important molecular components for biotechnological application and development of sustainable resistance against Foc1.

Functional analysis of the promoter of a glycosyl hydrolase gene induced in resistant Sinapis alba by Alternaria brassicicola.

A putative family 3 glycosyl hydrolase (GH) gene showed significant differential expression in resistant Sinapis alba, compared with the susceptible Brassica juncea, as part of the initial responses during interaction with the necrotroph Alternaria brassicicola. To understand the mechanism of induction, the promoter was isolated and deletion analysis carried out. All the promoter fragments were fused with the ß-glucuronidase gene and the expressions were studied in stable B. juncea transgenics and transiently transformed Nicotiana tabacum. Analysis of the expression of the promoter showed the presence of functional abscisic acid (ABA)-, jasmonic acid (JA)-, and salicylic acid (SA)-responsive cis elements. Interestingly, the promoter was found to be induced in both S. alba and B. juncea upon challenge with A. brassicicola but, in S. alba, SA had an inhibitory effect on the pathogen-induced expression of the gene whereas, in B. juncea, SA did not have any negative effect. Therefore, the SA-mediated inhibition in S. alba indicates that the induction is probably through JA or ABA signaling. The difference in the mechanism of induction of the same promoter in the resistant and susceptible plants is probably due to the differential hormonal responses initiated upon challenge with A. brassicicola.

The rolB-transgenic Nicotiana tabacum plants exhibit upregulated ARF7 and ARF19 gene expression.

Agrobacterium rhizogenes root oncogenic locus B (rolB) is known to induce hairy roots along with triggering several physiological and morphological changes when present as a transgene. However, it is still unknown how this gene triggers these changes within the plant system. In this study, the effect of rolB in-planta, when present as a transgene, was assessed on the gene expression levels of auxin response factors (ARFs)-transcription factors which are key players in auxin-mediated responses. The goal was to uncover Auxin/ARF-driven transcriptional networks potentially active and working selectively, if any, in rolB transgenic background, which might potentially be associated with hairy root development. Hence, the approach involved establishing rolB-transgenic Nicotiana tabacum plants, selecting ARFs (NtARFs) for context-relevance using bioinformatics followed by gene expression profiling. It was observed that out of the chosen NtARFs, NtARF7 and NtARF19 exhibited a consistent pattern of gene upregulation across organ types. In order to understand the significance of these selective gene upregulation, ontology-based transcriptional network maps of the differentially and nondifferentially expressed ARFs were constructed, guided by co-expression databases. The network maps suggested that NtARF7-NtARF19 might have major deterministic, underappreciated roles to play in root development in a rolB-transgenic background-as observed by higher number of "root-related" biological processes present as nodes compared to network maps for similarly constructed other non-differentially expressed ARFs. Based on the inferences drawn, it is hypothesized that rolB, when present as a transgene, might drive hairy root development by selective induction of NtARF7 and NtARF19, suggesting a functional link between the two, leading to the specialized and characteristic rolB-associated traits.

Inducible expression of truncated NAC62 provides tolerance against Alternaria brassicicola and imparts developmental changes in Indian mustard.

Indian mustard (Brassica juncea) faces significant yield loss due to the 'Black Spot Disease,' caused by a fungus Alternaria brassicicola. In plants, NAC transcription factors (NAC TFs) are known for their roles in development and stress tolerance. One such NAC TF, NAC 62, was induced during A. brassicicola challenge in Sinapis alba, a non-host resistant plant against this fungus. Sequence analyses of BjuNAC62 from B. juncea showed that it belonged to the membrane-bound class of transcription factors. Gene expression study revealed differential protein processing of NAC62 between B. juncea and S. alba on pathogen challenge. Furthermore, NAC62 processing to 25 kDa protein was found to be unique to the resistant plant during pathogenesis. Conditional expression of BjuNAC62ΔC, which lacks its transmembrane domain, in B. juncea showed improved tolerance to A. brassicicola. BjuNAC62ΔC processing to 25 kDa product was also observed in tolerant transgenic plants. Additionally, transgenic plants showed induced expression of genes associated with defense-related phytohormone signaling pathways on pathogen challenge. Again, altered phenotypes suggest a possible developmental effect of BjuNAC62∆C in transgenic plants. The overall results suggest that the processing of BjuNAC62 might be playing a crucial role in resistance response against Black Spot disease by modulating defense-associated genes.

Importance and results of sterility testing of various products in a microbiology laboratory of eastern India.

Components of blood products from Blood bank, stem cells products from Haemotapoietic Stem Cell Transplant unit, CSSD (Central Sterile Supply Department) items, and pharrrmaceutical products, were sterility tested by liquid culture. 2.91% of the total 3122 samples sent for sterility testing from various departments were positive (i.e. showing contamination). CSSD products showed no contamination (0/37); products from blood bank and bone marrow transplant unit showed a contamination rate of 2.03% (47/2307) and 4.64% (31/667) respectively. The average cost of sterility test was Rs. 302 (INR). Sterility test requires stringent aseptic precautions which is resource intensive.

Macro-to-micro porous special bioactive glass and ceftriaxone-sulbactam composite drug delivery system for treatment of chronic osteomyelitis: an investigation through in vitro and in vivo animal trial.

A systematic and extensive approach incorporating in vitro and in vivo experimentation to treat chronic osteomyelitis in animal model were made using antibiotic loaded special bioactive glass porous scaffolds. After thorough characterization for porosity, distribution, surface charge, a novel drug composite were infiltrated by using vacuum infiltration and freeze-drying method which was subsequently analyzed by SEM-EDAX and studied for in vitro drug elution in PBS and SBF. Osteomyelitis in rabbit was induced by inoculation of Staphylococcus aureus and optimum drug-scaffold were checked for its efficacy over control and parenteral treated animals in terms of histopathology, radiology, in vivo drug concentration in bone and serum and implant-bone interface by SEM. It was optimized that 60P samples with 60-65% porosity (bimodal distribution of macro- to micropore) with average pore size ~60 µm and higher interconnectivity, moderately high antibiotic adsorption efficiency (~49%) was ideal. Results after 42 days showed antibiotic released higher than MIC against S. aureus compared to parenteral treatment (2 injections a day for 6 weeks). In vivo drug pharmacokinetics and SEM on bone-defect interface proved superiority of CFS loaded porous bioactive glass implants over parenteral group based on infection eradication and new bone formation.

Local drug delivery system for the treatment of osteomyelitis: In vitro evaluation.

Local antimicrobial delivery is a potential area of research conceptualized to provide alternative and better methods of treatment for cases, as osteomyelitis where avascular zones prevent the delivery of drugs from conventional routes of administration. Drug-loaded polymers and calcium phosphates as hydroxyapatites have been tried earlier. Bioactive glasses are bone-filling materials used for space management in orthopedic and dental surgery. A new bioactive glass (SSS2) was synthesized and fabricated into porous scaffold with a view to provide prolonged local delivery of gatifloxacin and fluconazole as suitable for the treatment of osteomyelitis. The new SSS2 was characterized by Fourier transform infrared (FTIR) and X-ray diffraction (XRD) analyses. In addition, the bioactivity of the SSS2 glass and resulting scaffold was examined by in vitro acellular method and ascertained by FTIR and XRD. The pore size distribution was analysed by mercury intrusion porosimetry and the release of drugs from scaffolds were studied in vitro. The glass and the resulting scaffolds were bioactive indicating that they can bond with bone in vivo. The scaffolds were porous with pores predominantly in the range of 10-60 µm, released the drugs effectively for 6 weeks and deemed suitable for local delivery of drugs to treat osteomyelitis.

Overexpression of LYK4, a lysin motif receptor with non-functional kinase domain, enhances tolerance to Alternaria brassicicola and increases trichome density in Brassica juncea.

Lysin motif receptor-like kinases (LYKs) are involved in the recognition of chitin and activation of plant immune response. In this study, we found LYK4 to be strongly induced in resistant Sinapis alba compared with susceptible Brassica juncea on challenge with Alternaria brassicicola. In silico analysis and in vitro kinase assay revealed that despite the presence of canonical protein kinase fold, B.juncea LYK4 (BjLYK4) lacks several key residues of a prototype protein kinase which renders it catalytically inactive. Transient expression analysis confirmed that fluorescently tagged BjLYK4 localizes specifically to the plasma membrane. Overexpression (OE) of BjLYK4 in B. juncea enhanced tolerance against A. brassicicola. Interestingly, the OE lines also exhibited a novel trichome dense phenotype and increased jasmonic acid (JA) responsiveness. We further showed that many chitin responsive WRKY transcription factors and JA biosynthetic genes were strongly induced in the OE lines on challenge with the pathogen. Moreover, several JA inducible trichome developmental genes constituting the WD-repeat/bHLH/MYB activator complex were also upregulated in the OE lines compared with vector control and RNA interference line. These results suggest that BjLYK4 plays an essential role in chitin-dependent activation of defense response and chitin independent trichome development likely by influencing the JA signaling pathway.

An unedited 1.1 kb mitochondrial orfB gene transcript in the wild abortive cytoplasmic male sterility (WA-CMS) system of Oryza sativa L. subsp. indica.

BACKGROUND: The application of hybrid rice technology has significantly increased global rice production during the last three decades. Approximately 90% of the commercially cultivated rice hybrids have been derived through three-line breeding involving the use of WA-CMS lines. It is believed that during the 21st century, hybrid rice technology will make significant contributions to ensure global food security. This study examined the poorly understood molecular basis of the WA-CMS system in rice. RESULTS: RFLPs were detected for atp6 and orfB genes in sterile and fertile rice lines, with one copy of each in the mt-genome. The RNA profile was identical in both lines for atp6, but an additional longer orfB transcript was identified in sterile lines. 5' RACE analysis of the long orfB transcript revealed it was 370 bp longer than the normal transcript, with no indication it was chimeric when compared to the genomic DNA sequence. cDNA clones of the longer orfB transcript in sterile lines were sequenced and the transcript was determined unedited. Sterile lines were crossed with the restorer and maintainer lines, and fertile and sterile F1 hybrids were respectively generated. Both hybrids contained two types of orfB transcripts. However, the long transcript underwent editing in the fertile F1 hybrids and remained unedited in the sterile lines. Additionally, the editing of the 1.1 kb orfB transcript co-segregated with fertility restoring alleles in a segregating population of F2 progeny; and the presence of unedited long orfB transcripts was detected in the sterile plants from the F2 segregating population. CONCLUSION: This study helped to assign plausible operative factors responsible for male-sterility in the WA cytoplasm of rice. A new point of departure to dissect the mechanisms governing the CMS-WA system in rice has been identified, which can be applied to further harness the opportunities afforded by hybrid vigor in rice.

Development of new localized drug delivery system based on ceftriaxone-sulbactam composite drug impregnated porous hydroxyapatite: a systematic approach for in vitro and in vivo animal trial.

PURPOSE: Present investigation deals with an extensive approach incorporating in vitro and in vivo experimentation to treat chronic osteomyelitis, using hydroxyapatite porous scaffolds. MATERIALS AND METHODS: Hydroxyapatite was synthesized in the laboratory by wet chemical method, different porous scaffolds have been fabricated. In vitro studies include variation of porosity with interconnectivity, pore-drug interfacial studies by SEM-EDAX and drug elution studies (by HPLC) both in contact with PBS and SBF at approximately 37 degrees C. In vivo trials were based on experimental osteomyelitis in rabbit model induced in tibia by Staphylococcus aureus. Characterizations included observation of histopathology, radiology and estimation of drug in both bone and serum for 42 days by HPLC method and subsequent bone-biomaterial interface by SEM. RESULTS: It was established that lower pore percentage with a distribution of mainly micro-pores were found to be superior over the higher pore percentage both in vitro and in vivo. The criteria was matched with the 50N50H samples which had 50-55% porosity with an average pore size approximately 110 microm, having higher interconnectivity (10-100 microm), moderately high adsorption efficiency (approximately 50%) when loaded with CFS (drug combinations consisting of irreversible b-lactamase inhibitor and b-lactam antibiotic). CFS release from HAp implants were faster in PBS than SBF. Further, both the results of in vitro and in vivo drug elution after 42 days showed release higher than minimum inhibitory concentration of CFS against Staphylococcus aureus. In vivo studies also proved the superiority of CFS loaded HAp implants than parenteral group based on eradication of infection and new bone formation. CONCLUSIONS: HAp based porous scaffold loaded with CFS and designed porosity (in terms of micro- and macro-porosity, interconnectivity) was found to be an ideal delivery system which could locally, sustainably release the composite antibiotic in reliable manner both in terms of in vitro drug elution behaviour in contact with SBF and in vivo animal trial.

Porous bioactive glass scaffolds for local drug delivery in osteomyelitis: development and in vitro characterization.

A new bioactive glass-based scaffold was developed for local delivery of drugs in case of osteomyelitis. Bioactive glass having a new composition was prepared and converted into porous scaffold. The bioactivity of the resulting scaffold was examined by in vitro acellular method. The scaffolds were loaded with two different drugs, an antibacterial or antifungal drug. The effects of the size of the scaffold, drug concentration, and dissolution medium on drug release were studied. The scaffolds were further coated with a degradable natural polymer, chitosan, to further control the drug release. Both the glass and scaffold were bioactive. The scaffolds released both the drugs for 6 weeks, in vitro. The results indicated that the bigger the size and the higher the drug concentration, the better was the release profile. The scaffolds appeared to be suitable for local delivery of the drugs in cases of osteomyelitis.

A molecular insight into the early events of chickpea (Cicer arietinum) and Fusarium oxysporum f. sp. ciceri (race 1) interaction through cDNA-AFLP analysis.

Wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris is one of the most severe diseases of chickpea throughout the world. Variability of pathotypes of F. oxysporum f. sp. ciceris and breakdown of natural resistance are the main hindrances to developing resistant plants by applying resistant breeding strategies. Additionally, lack of information of potential resistant genes limits gene-transfer technology. A thorough understanding of Fusarium spp.-chickpea interaction at a cellular and molecular level is essential for isolation of potential genes involved in counteracting disease progression. Experiments were designed to trigger the pathogen-challenged disease responses in both susceptible and resistant plants and monitor the expression of stress induced genes or gene fragments at the transcript level. cDNA amplified fragment length polymorphism followed by homology search helped in differentiating and analyzing the up- and downregulated gene fragments. Several detected DNA fragments appeared to have relevance with pathogen-mediated defense. Some of the important transcript-derived fragments were homologous to genes for sucrose synthase, isoflavonoid biosynthesis, drought stress response, serine threonine kinases, cystatins, arginase, and so on. Reverse-transcriptase polymerase chain reaction performed with samples collected at 48 and 96 h postinfection confirmed a similar type of differential expression pattern. Based on these results, interacting pathways of cellular processes were generated. This study has an implication toward functional identification of genes involved in wilt resistance.

The repair of segmental bone defects with porous bioglass: an experimental study in goat.

This study was exclusively conducted to evaluate healing of surgically created defects on the radius of adult Black Bengal goat after implantation of porous bioglass blocks and compare the process kinetics with normal healing. Twelve Black Bengal goats were divided randomly into two groups: control and experimental group implanted with bioglass blocks. Unicortical bone defects in radius were generated in all animals under aseptic condition. Local inflammatory reaction and healing of wound, radiological investigations, histological studies, oxytetracycline leveling and angiographic studies were performed up to 90th day post-operatively and compared with normal healing. It has been found that extensive new bone formation originating from host bone towards the implant whereas in control, the process was active from both the ends; the defect site appeared as homogenous nonfluorescent area. Thus, porous bioglass promoted bone formation over the entire extension of the defect independent of size of block in comparison to control group.

Self-assembled structures of hydroxyapatite in the biomimetic coating on a bioinert ceramic substrate.

The tribological properties of alumina ceramic are excellent due in part to a high wettability because of the hydrophilic surface and fluid film lubrication that minimizes the adhesive wear. Such surfaces are further modified with bioactive glass/ceramic coating to promote direct bone apposition in orthopedic applications. The present communication reports the biomimetic coating of calcium hydroxyapatite (HAp) on dense (2-3% porosity) alumina (alpha-Al(2)O(3)) substrate (1cm x 1cm x 0.5 cm), at 37 degrees C. After a total period of 6 days immersion in simulated body fluid (SBF), at 37 degrees C, linear self-assembled porous (pore size: approximately 0.2 microm) structures (length: approximately 375.39 microm and width: 5-6 microm) of HAp were obtained. The phenomenon has been demonstrated by self-assembly and diffusion-limited aggregation (DLA) principles. Structural and compositional characterization of the coating was carried out using SEM with EDX facility, XRD and FT-IR data.

Efficacy of nano-hydroxyapatite prepared by an aqueous solution combustion technique in healing bone defects of goat.

The present study was undertaken to evaluate porous hydroxyapatite (HAp), the powder of which was prepared by a novel aqueous solution combustion technique, as a bone substitute in healing bone defects in vivo, as assessed by radiologic and histopathologic methods, oxytetracycline labeling, and angiogenic features in Bengal goat. Bone defects were created in the diaphysis of the radius and either not filled (group I) or filled with a HAp strut (group II). The radiologic study in group II showed the presence of unabsorbed implants which acted as a scaffold for new bone growth across the defect, and the quality of healing of the bone defect was almost indistinguishable from the control group, in which the defect was more or less similar, although the newly formed bony tissue was more organized when HAp was used. Histologic methods showed complete normal ossification with development of Haversian canals and well-defined osteoblasts at the periphery in group II, whereas the control group had moderate fibro-collagenization and an adequate amount of marrow material, fat cells, and blood vessels. An oxytetracycline labeling study showed moderate activity of new bone formation with crossing-over of new bone trabeculae along with the presence of resorption cavities in group II, whereas in the control group, the process of new bone formation was active from both ends and the defect site appeared as a homogenous non-fluoroscent area. Angiograms of the animals in the control group showed uniform angiogenesis in the defect site with establishment of trans-transplant angiogenesis, whereas in group II there was complete trans-transplant shunting of blood vessel communication. Porous HAp ceramic prepared by an aqueous combustion technique promoted bone formation over the defect, confirming their biologic osteoconductive property.

Reason behind wet pack after steam sterilization and its consequences: An overview from Central Sterile Supply Department of a cancer center in eastern India.

Wet pack after steam sterilization process that means there are surely obtain millions of microorganisms that can breed and multiply rapidly and objects are unsterile and can never be used for further procedure. There are many reasons behind the wet pack occurrences after autoclaving like poor quality of wrapping materials, faulty valves of rigid container, faulty loading and packaging technique, poor steam quality, sterilizer malfunction and may be design related problems in CSSD sterile storage area. Cause of wet pack after steam sterilization processes may occur severe problems because of wasted time and effort, increased work load, increased cost, potentially contaminated instruments, infection risk to the patient, poor patient outcomes and delayed or cancellation of procedures. But such wet pack scenario can be avoided by various methods by using good steam (water) quality, performing periodic maintenance of the Autoclaves, avoidance of sterilizer overloading, allowing adequate post sterilization time to cool down the materials to room temperature, using good quality wrapping materials, properly maintain temperature and humidity of sterile storage area etc.