IND-enzymes: a repository for hydrolytic enzymes derived from thermophilic and psychrophilic bacterial species with potential industrial usage.

Biocatalysts provide many advantages over the traditional chemically assisted processes prevalent in industries. Consequently, the search for novel enzymes has increased over the years with a renewed interest in thermophilic and psychrophilic bacterial species. Enzymes or extremozymes extracted from such species have exhibited an affinity to extreme temperatures which is a prerequisite for many industrial applications. However, utilisation of these enzymes faces a major bottleneck. The distribution of sequence data associated with thermophiles and psychrophiles is overwhelming, spanning various databases and scientific literature. Based on more than 100 publications and genomes from over 300 thermophilic and psychrophilic bacterial species, we have constructed the database IND-Enzymes (indenzymes.srmist.edu.in). This database consists of over 20,120 nucleotide and protein sequences belonging to the hydrolytic enzyme class lipase, protease, esterase and amylase. Users can access over 100 published enzymes, 200 PDB structural data. Enzymes derived from genomes can be directly downloaded and users can also access the entire annotation data derived from species individually. Along with an alignment tool and python based pipelines, IND-Enzymes serves as the largest sequence repository for hydrolytic enzymes from thermophilic and psychrophilic bacterial species. This database showcases resources that are essential for protein engineering of hot-cold stable enzymes.

Compost Samples from Different Temperature Zones as a Model to Study Co-occurrence of Thermophilic and Psychrophilic Bacterial Population: a Metagenomics Approach.

In this study, using a metagenomic approach, we explore the bacterial diversity of compost sites categorized based on their ambient temperatures. The two sites were Reckong Peo in the lower Himalayas and Tambaram in the southern region of the country, namely, CPR and CT. Following assembly of the raw reads from shotgun metagenomics, similarity hits were generated using NCBI BLAST + and SILVA database. A total of 1463 and 1483 species were annotated from CPR and CT. A species-level annotation was performed using a python-based literature search pipeline revealing their growth characteristics. Thermophiles Thermomonospora curvata and Thermus scotoductus were among the prominent species in CT. CPR too was seen abundant with Acidothermus cellulolyticus and Moorella thermoacetica, constituting 10% of the population. Nearly 3% of the identified species in the site CPR were psychrophilic. Although found higher in CPR, psychrophilic species were identified in CT too. Flavobacterium and Psychrobacter spp. were present in both sites without any significant changes in their relative distribution contrary to the thermophilic species abundance (z = - 4.3). Akin to the sequenced samples, database-derived metagenomes also showed similar distribution of thermophiles and psychrophiles. Identifying such peculiar prevalence of extremophiles can be central to understanding extended growth temperatures.

Identifying heat shock response systems from the genomic assembly of Ureibacillus thermophilus LM102 using protein-protein interaction networks.

Ureibacillus thermophilus strain LM102 is a facultative thermophile with growth in range 37 °C-60 °C. Upon identification using the 16S rRNA marker, it showed highest similarity of 99.8% with U. thermophilus strain HC148. A phylogenetic analysis revealed U. suwonensis strain 6 T19 to be the closest species to strain LM102. Our aim was to determine the unique thermotolerant properties of LM102 by identifying thermostablity of its proteins and the interactions existing in its heat shock response systems (HSRS). The strain was sequenced, assembled and the draft genome (3,017,325 bp) was analyzed. Post-annotation, we randomly selected a set of hundred proteins and computed the percentage distribution of 12 amino acids which have been substantially studied for their role in thermostability. The protein homologues were searched and the residues of LM102 were compared with Bacillus subtilis and Thermus thermophilus, a mesophile and hyperthermophile respectively. Within the 95% confidence limit, a Z-score of -0.61 was observed between LM102 and B. subtilis. However, a significantly lower value of -8.84 was observed for the pair LM102 and T. thermophilus. The amino acid distribution did not appear to influence the protein thermostability. Further, we investigated the role of Protein-Protein interactions by building networks for heat shock responses, namely DNA repair, transcriptional regulation, and activation of heat shock proteins. Interaction data retrieved from the STRING database for more than 50 species were used to build these networks. Highly clustered MCODE results notably revealed RNA 3'-5' exonuclease, CshA and HemW previously unreported, in association with other proteins. Additionally, these and other proteins estimated from the HSRS networks were found in both mesophiles and thermophiles, suggesting a crucial role of gene regulatory networks in the cellular viability of LM102 at high temperatures.

Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors.

Depending on the inflammatory milieu, injury can result either in a tissue's complete regeneration or in its degeneration and fibrosis, the latter of which could potentially lead to permanent organ failure. Yet how inflammatory cells regulate matrix-producing cells involved in the reparative process is unknown. Here we show that in acutely damaged skeletal muscle, sequential interactions between multipotent mesenchymal progenitors and infiltrating inflammatory cells determine the outcome of the reparative process. We found that infiltrating inflammatory macrophages, through their expression of tumor necrosis factor (TNF), directly induce apoptosis of fibro/adipogenic progenitors (FAPs). In states of chronic damage, however, such as those in mdx mice, macrophages express high levels of transforming growth factor ß1 (TGF-ß1), which prevents the apoptosis of FAPs and induces their differentiation into matrix-producing cells. Treatment with nilotinib, a kinase inhibitor with proposed anti-fibrotic activity, can block the effect of TGF-ß1 and reduce muscle fibrosis in mdx mice. Our findings reveal an unexpected anti-fibrotic role of TNF and suggest that disruption of the precisely timed progression from a TNF-rich to a TGF-ß-rich environment favors fibrotic degeneration of the muscle during chronic injury.

EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation.

Hepatocellular carcinoma (HCC) is a frequent cancer with limited treatment options and poor prognosis. Tumorigenesis has been linked with macrophage-mediated chronic inflammation and diverse signalling pathways, including the epidermal growth factor receptor (EGFR) pathway. The precise role of EGFR in HCC is unknown, and EGFR inhibitors have shown disappointing clinical results. Here we discover that EGFR is expressed in liver macrophages in both human HCC and in a mouse HCC model. Mice lacking EGFR in macrophages show impaired hepatocarcinogenesis, whereas mice lacking EGFR in hepatocytes unexpectedly develop more HCC owing to increased hepatocyte damage and compensatory proliferation. Mechanistically, following interleukin-1 stimulation, EGFR is required in liver macrophages to transcriptionally induce interleukin-6, which triggers hepatocyte proliferation and HCC. Importantly, the presence of EGFR-positive liver macrophages in HCC patients is associated with poor survival. This study demonstrates a tumour-promoting mechanism for EGFR in non-tumour cells, which could lead to more effective precision medicine strategies.

Nonmyogenic cells in skeletal muscle regeneration.

Although classical dogma dictates that satellite cells are the primary cell type involved in skeletal muscle regeneration, alternative cell types such as a variety of inflammatory and stromal cells are also actively involved in this process. A model describing myogenic cells as direct contributors to regeneration and nonmyogenic cells from other developmental sources as important accessories has emerged, with similar systems having been described in numerous other tissues in the body. Increasing evidence supports the notion that inflammatory cells function as supportive accessory cells, and are not merely involved in clearing damage following skeletal muscle injury. Additionally, recent studies have highlighted the role of tissue resident mesenchymal cell populations as playing a central role in regulating regeneration. These "accessory" cell populations are proposed to influence myogenesis via direct cell contact and secretion of paracrine trophic factors. The basic foundations of accessory cell understanding should be recognized as a crucial component to all prospects of regenerative medicine, and this chapter intends to provide a comprehensive background on the current literature describing immune and tissue-resident mesenchymal cells' role in skeletal muscle regeneration.

Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.

Efficient tissue regeneration is dependent on the coordinated responses of multiple cell types. Here, we describe a new subpopulation of fibro/adipogenic progenitors (FAPs) resident in muscle tissue but arising from a distinct developmental lineage. Transplantation of purified FAPs results in the generation of ectopic white fat when delivered subcutaneously or intramuscularly in a model of fatty infiltration, but not in healthy muscle, suggesting that the environment controls their engraftment. These cells are quiescent in intact muscle but proliferate efficiently in response to damage. FAPs do not generate myofibres, but enhance the rate of differentiation of primary myogenic progenitors in co-cultivation experiments. In summary, FAPs expand upon damage to provide a transient source of pro-differentiation signals for proliferating myogenic progenitors.

The epidermal growth factor receptor: from development to tumorigenesis.

The epidermal growth factor receptor (EGFR) is activated by many ligands and belongs to a family of tyrosine kinase receptors, including ErbB2, ErbB3, and ErbB4. These receptors are de-regulated in many human tumors, and EGFR amplification, overexpression, and mutations are detected at a high frequency in carcinomas and glioblastomas, which are tumors of epithelial and glial origin, respectively. From the analysis of EGFR-deficient mice, it seems that the cell types mostly affected by the absence of EGFR are epithelial and glial cells, the same cell types where the EGFR is found to be overexpressed in human tumors. Therefore, it is important to define molecularly the function of EGFR signaling in the development of these cell types, because this knowledge will be of fundamental importance to understand how aberrant EGFR signaling can lead to tumor formation and progression. A molecular understanding of the pathways that control the development of a given tissue or cell type will also provide the basis for developing better combination therapies targeting different key components of the EGFR signaling network in the respective cancerous cells. Here, we will review the current knowledge, mostly derived from the analysis of genetically modified mice and cells, about the function of the EGFR in specific organs and tissues and in sites where the EGFR is found to be overexpressed in human tumors.

The EGF receptor is required for efficient liver regeneration.

Mice lacking the EGF receptor (EGFR) die between midgestation and postnatal day 20 with various defects in neural and epithelial organs. Here, we generated mice carrying a floxed EGFR allele to inactivate the EGFR in fetal and adult liver. Perinatal deletion of EGFR in hepatocytes resulted in decreased body weight, whereas deletion in the adult liver did not affect body mass. Although liver function was not affected, after partial hepatectomy mice lacking EGFR in the liver showed increased mortality accompanied by increased levels of serum transaminases indicating liver damage. Liver regeneration was delayed in the mutants because of reduced hepatocyte proliferation. Analysis of cell cycle progression in EGFR-deficient livers indicated a defective G(1)-S phase entry with delayed transcriptional activation and reduced protein expression of cyclin D1 followed by reduced cdk2 and cdk1 expression. Impaired liver regeneration was accompanied by compensatory up-regulation of TNFalpha in the serum and prolonged activation of c-Jun. Moreover, p38alpha and NF-kappaB activation was reduced in regenerating mutant livers, indicating an impaired stress response after hepatectomy. Our studies demonstrate that EGFR is a critical regulator of hepatocyte proliferation in the initial phases of liver regeneration.

Neuronal survival depends on EGFR signaling in cortical but not midbrain astrocytes.

Mice lacking epidermal growth factor receptor (EGFR) develop a neurodegeneration of unknown etiology affecting exclusively the frontal cortex and olfactory bulbs. Here, we show that EGFR signaling controls cortical degeneration by regulating cortical astrocyte apoptosis. Whereas EGFR(-/-) midbrain astrocytes are unaffected, mutant cortical astrocytes display increased apoptosis mediated by an Akt-caspase-dependent mechanism and are unable to support neuronal survival. The expression of many neurotrophic factors is unaltered in EGFR(-/-) cortical astrocytes suggesting that neuronal loss occurs as a consequence of increased astrocyte apoptosis rather than impaired secretion of trophic factors. Neuron-specific expression of activated Ras can compensate for the deficiency of EGFR(-/-) cortical astrocytes and prevent neuronal death. These results identify two functionally distinct astrocyte populations, which differentially depend on EGFR signaling for their survival and also for their ability to support neuronal survival. These spatial differences in astrocyte composition provide a mechanism for the region-specific neurodegeneration in EGFR(-/-) mice.