Unraveling the complex regulatory networks in biofilm formation in bacteria and relevance of biofilms in environmental remediation.

Biofilms are assemblages of bacteria embedded within a matrix of extracellular polymeric substances (EPS) attached to a substratum. The process of biofilm formation is a complex phenomenon regulated by the intracellular and intercellular signaling systems. Various secondary messenger molecules such as cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP), cyclic adenosine 3',5'-monophosphate (cAMP), and cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) are involved in complex signaling networks to regulate biofilm development in several bacteria. Moreover, the cell to cell communication system known as Quorum Sensing (QS) also regulates biofilm formation via diverse mechanisms in various bacterial species. Bacteria often switch to the biofilm lifestyle in the presence of toxic pollutants to improve their survivability. Bacteria within a biofilm possess several advantages with regard to the degradation of harmful pollutants, such as increased protection within the biofilm to resist the toxic pollutants, synthesis of extracellular polymeric substances (EPS) that helps in the sequestration of pollutants, elevated catabolic gene expression within the biofilm microenvironment, higher cell density possessing a large pool of genetic resources, adhesion ability to a wide range of substrata, and metabolic heterogeneity. Therefore, a comprehensive account of the various factors regulating biofilm development would provide valuable insights to modulate biofilm formation for improved bioremediation practices. This review summarizes the complex regulatory networks that influence biofilm development in bacteria, with a major focus on the applications of bacterial biofilms for environmental restoration.

Electroactive biofilm communities in microbial fuel cells for the synergistic treatment of wastewater and bioelectricity generation.

Increasing industrialization and urbanization have contributed to a significant rise in wastewater discharge and exerted extensive pressure on the existing natural energy resources. Microbial fuel cell (MFC) is a sustainable technology that utilizes wastewater for electricity generation. MFC comprises a bioelectrochemical system employing electroactive biofilms of several aerobic and anaerobic bacteria, such as Geobacter sulfurreducens, Shewanella oneidensis, Pseudomonas aeruginosa, and Ochrobacterum pseudiintermedium. Since the electroactive biofilms constitute a vital part of the MFC, it is crucial to understand the biofilm-mediated pollutant metabolism and electron transfer mechanisms. Engineering electroactive biofilm communities for improved biofilm formation and extracellular polymeric substances (EPS) secretion can positively impact the bioelectrochemical system and improve fuel cell performance. This review article summarizes the role of electroactive bacterial communities in MFC for wastewater treatment and bioelectricity generation. A significant focus has been laid on understanding the composition, structure, and function of electroactive biofilms in MFC. Various electron transport mechanisms, including direct electron transfer (DET), indirect electron transfer (IET), and long-distance electron transfer (LDET), have been discussed. A detailed summary of the optimization of process parameters and genetic engineering strategies for improving the performance of MFC has been provided. Lastly, the applications of MFC for wastewater treatment, bioelectricity generation, and biosensor development have been reviewed.

Cellulolytic potential of mangrove bacteria Bacillus haynesii DS7010 and the effect of anthropogenic and environmental stressors on bacterial survivability and cellulose metabolism.

Cellulose degrading bacterial diversity of Bhitarkanika mangrove ecosystem, India, was uncovered and the cellulose degradation mechanism in Bacillus haynesii DS7010 under the modifiers such as pH (pCO2), salinity and lead (Pb) was elucidated in the present study. The abundance of cellulose degrading heterotrophic bacteria was found to be higher in mangrove sediment than in water. The most potential strain, B. haynesii DS7010 showed the presence of endoglucanase, exoglucanase and ß-glucosidase with the maximum degradation recorded at 48 h of incubation, with 1% substrate concentration at 41 °C incubation temperature. Two glycoside hydrolase genes, celA and celB were confirmed in this bacterium. 3D structure prediction of the translated CelA and CelB proteins showed maximum similarities with glycoside hydrolase 48 (GH48) and glycoside hydrolase 5 (GH5) respectively. Native PAGE followed by zymogram assay unveiled the presence of eight isoforms of cellulase ranged from 78 kDa to 245 kDa. Among the stressors, most adverse effect was observed under Pb stress at 1400 ppm concentration, followed by pH at pH 4. This was indicated by prolonged lag phase growth, higher reactive oxygen species (ROS) production, lower enzyme activity and downregulation of celA and celB gene expressions. Salinity augmented bacterial metabolism up to 3% NaCl concentration. Mangrove leaf litter degradation by B. haynesii DS7010 indicated a substantial reduction in cellulolytic potential of the bacterium in response to the synergistic effect of the stressors. Microcosm set up with the stressors exhibited 0.97% decrease in total carbon (C%) and 0.02% increase in total nitrogen (N%) after 35 d of degradation while under natural conditions, the reduction in C and the increase in N were 4.05% and 0.2%, respectively. The findings of the study suggest the cellulose degradation mechanism of a mangrove bacterium and its resilience to the future consequences of environmental pollution and climate change.

A novel rspA gene regulates biofilm formation and virulence of Salmonella Typhimurium.

Salmonella spp. are facultative anaerobic, Gram-negative, rod-shaped bacteria and belongs to the Enterobacteriaceae family. Although much has been known about Salmonella pathogenesis, the functional characterizations of certain genes are yet to be explored. The rspA (STM14_1818) is one such gene with putative dehydratase function, and its role in pathogenesis is unknown. The background information showed that rspA gene is upregulated in Salmonella when it resides inside macrophages, which led us to investigate its role in Salmonella pathogenesis. We generated the rspA knockout strain and complement strain in S. Typhimurium 14028. Ex-vivo and in-vivo infectivity was looked at macrophage and epithelial cell lines and Caenorhabditis elegans (C. elegans). The mutant strain differentially formed the biofilm at different temperatures by altering the expression of genes involved in the synthesis of cellulose and curli. Besides, the mutant strain is hyperproliferative intracellularly and showed increased bacterial burden in C. elegans. The mutant strain became more infectious and lethal, causing faster death of the worms than the wild type, and also modulates the worm's innate immunity. Thus, we found that the rspA deletion mutant was more pathogenic. In this study, we concluded that the rspA gene differentially regulates the biofilm formation in a temperature dependent manner by modulating the genes involved in the synthesis of cellulose and curli and negatively regulates the Salmonella virulence for longer persistence inside the host.

Concentration- and Solvent-Induced Chiral Tuning by Manipulating Non-Proteinogenic Amino Acids in Glycoconjugate Supra-Scaffolds: Interaction with Protein, and Streptomycin Delivery.

We showed solvent- and concentration-triggered chiral tuning of the fibrous assemblies of two novel glycoconjugates Z-P(Gly)-Glu and Z-F(4-N)-Glu made by chemical attachment of Cbz-protected [short as Z)] non-proteinogenic amino acids L-phenylglycine [short as P(Gly)] and 4-Nitro-L-phenylalanine [short as F(4-N)] with D-glucosamine [short as Glu]. Both biomimetic gelators can form self-healing and shape-persistent gels with a very low critical gelator concentration in water as well as in various organic solvents, indicating they are ambidextrous supergelators. Detailed spectroscopic studies suggested ß-sheet secondary structure formation during anisotropic self-aggregation of the gelators which resulted in the formation of hierarchical left-handed helical fibers in acetone with an interlayer spacing of 2.4 nm. After the physical characterization of the gels, serum protein interaction with the gelators was assessed, indicating they may be ideal for biomedical applications. Further, both gelators are benign, non-immunogenic, non-allergenic, and non-toxic in nature, which was confirmed by performing the blood parameters and liver function tests on Wister rats. Streptomycin-loaded hydrogels showed efficacious antibacterial activity in vitro and in vivo as well. Finally, cell attachment and biocompatibility of the hydrogels were demonstrated which opens a newer avenue for promising biomedical and therapeutic applications.

Formulation and Skin Permeation of Active-Loaded Lipid Nanoparticles: Evaluation and Screening by Synergizing Molecular Dynamics Simulations and Experiments.

Encapsulation into nanoparticles (NPs) is a potential method to deliver pharmaceutical/cosmetic actives deep into the skin. However, understanding the NP formulations and underlying mechanism of active delivery to skin has scarcely been studied. We report a simulation platform that screens, evaluates, formulates, and provides atomic-resolution interpretation of NP-based formulations, and reveals the active permeation mechanism from NPs to skin. First, three actives, namely, ferulic acid (FA), clotrimazole (CZE), and tretinoin (TTN), and five lipid excipients' (Compritol, Precirol, Geleol, Gelot, Gelucire) combinations were screened by MD simulations for the best pairs. For each suggested pair, the actual active and lipid compositions for the synthesis of stable NP formulations were then obtained by experiments. MD simulations demonstrate that in NP formulations, FA and CZE actives are present at the surface of the NPs, whereas TTN actives are present at both the surface and interior of the NP core. The NP shapes obtained by simulation perfectly match with experiments. For each NP, separate MD simulations illustrate that active-loaded NPs approach the skin surface quickly, and then actives translocate from NP surface to skin surface followed by penetration of NPs through skin. The driving force for the translocation which initiates during the penetration process, is the stronger active-skin interaction compared to active-NP interaction. Permeation free energy indicates spontaneous transfer of actives from solution phase to the surface of the skin bilayer. The free energy barriers are increased in the order of FA < TTN < CZE. Significantly lower diffusions of actives are obtained in the main barrier region compared to bulk, and the average diffusion coefficients of actives are in the same order of magnitude (∼10-6 cm2/s). The estimated permeability coefficients (log P) of actives are mainly governed by free energy barriers. The study would facilitate the development of novel lipid-based NP formulations for personal-care/pharmaceutical applications.

Acid-tolerant bacteria and prospects in industrial and environmental applications.

Acid-tolerant bacteria such as Streptococcus mutans, Acidobacterium capsulatum, Escherichia coli, and Propionibacterium acidipropionici have developed several survival mechanisms to sustain themselves in various acid stress conditions. Some bacteria survive by minor changes in the environmental pH. In contrast, few others adapt different acid tolerance mechanisms, including amino acid decarboxylase acid resistance systems, mainly glutamate-dependent acid resistance (GDAR) and arginine-dependent acid resistance (ADAR) systems. The cellular mechanisms of acid tolerance include cell membrane alteration in Acidithiobacillus thioxidans, proton elimination by F1-F0-ATPase in Streptococcus pyogenes, biofilm formation in Pseudomonas aeruginosa, cytoplasmic urease activity in Streptococcus mutans, synthesis of the protective cloud of ammonia, and protection or repair of macromolecules in Bacillus caldontenax. Apart from cellular mechanisms, there are several acid-tolerant genes such as gadA, gadB, adiA, adiC, cadA, cadB, cadC, speF, and potE that help the bacteria to tolerate the acidic environment. This acid tolerance behavior provides new and broad prospects for different industrial applications and the bioremediation of environmental pollutants. The development of engineered strains with acid-tolerant genes may improve the efficiency of the transgenic bacteria in the treatment of acidic industrial effluents. KEY POINTS: • Bacteria tolerate the acidic stress by methylating unsaturated phospholipid tail • The activity of decarboxylase systems for acid tolerance depends on pH • Genetic manipulation of acid-tolerant genes improves acid tolerance by the bacteria.

Precision technologies for the management of reproduction in dairy cows.

Precision livestock farming (PLF) utilizes information and communication technology (ICT) to continuously monitor, control, and enhance the productivity, reproduction, health, welfare, and environmental impact of livestock. Technological advancements have facilitated the seamless flow of information from animals to humans, enabling practical decision-making processes concerning health, reproduction management, and calving surveillance. With the increasing population of livestock per farm, it has become impractical for farmers to individually track every animal within these large groups. Historically, cattle management decisions heavily relied on human observation, judgment, and experience. However, it is impossible for a single individual to gather reliable audio-visual monitoring data round the clock. Presently, dairy cows exhibit subtler indicators of estrus, resulting in a substantial chance of missing an estrus cycle. Furthermore, calving complications sometimes go unnoticed on farms, resulting in a higher number of culled cattle. In addition, an increasing number of crossbred cows experience delayed return to estrus after calving due to low body condition scores (BCS). The decline in BCS during the dry period is associated with a reduced likelihood of pregnancy following the first and second postpartum inseminations. Precision technologies enable the monitoring and tracking of an individual cow's physiological behavior and reproductive parameters, thereby optimizing management practices and farm performance. Despite the exploration of various technologies, there are still some common challenges that need to be addressed, including battery lifespan, transmission range, specificity and sensitivity, storage capacity, and economic affordability. Nonetheless, the demand for these tools from farmers and researchers is growing, and the implementation of PLF in grazing systems can yield positive outcomes in terms of animal reproductive welfare and labor optimization. This review primarily focuses on the different aspects of reproduction management in dairy using sensors, automated cameras, and various computer software.

Microbial community signatures for estimation of postmortem time intervals.

The human body provides a complex ecosystem for symbiotic habitation of a huge number of microorganisms. These commensal microorganisms provide a huge benefit to the living host by acting against many deadly infections. Once the host dies, many changes in the complex ecosystem of the human body take place. The personalized microbes of a human body undergo successional change as many exogenous microbes attack the nutrient-rich cadaver after death. The succession pattern change of microbes in human cadaver allows postulating different models for estimation of Postmortem time interval (PMI). Estimation of PMI has a broad prospect from the criminal investigation point of view. Though many techniques are being used nowadays to estimate PMI, all of them have their pros and cons. With the advent of advanced molecular biological techniques, studies on the thanatomicrobiome of a human cadaver have gained pace and provide a superior alternative for conventional methods of PMI estimation. This chapter summarizes the recent advancements in the changes in signature microflora postmortem with change in human microenvironment to postulate a consensus model for estimation of PMI.

Variable pH and subsequent change in pCO2 modulates the biofilm formation, synthesis of extracellular polymeric substances, and survivability of a marine bacterium Bacillus stercoris GST-03.

Biofilm-forming bacteria adhere to the substrates and engage in the nutrient cycling process. However, environmental conditions may interrupt the biofilm formation ability, which ultimately may affect various biogeochemical cycles. The present study reports the effect of varying pH and subsequent change in pCO2 on the survivability, biofilm formation, and synthesis of extracellular polymeric substances (EPS) of a biofilm-forming marine bacterium Bacillus stercoris GST-03 isolated from the Bhitarkanika mangrove ecosystem, Odisha, India. Understanding the pH-dependent alteration in EPS constituents, and associated functional groups of a marine bacterium will provide better insight into the adaptability of the bacteria in future ocean acidification scenarios. The strain was found to tolerate and form biofilm up to pH 4, with the maximum biofilm formation at pH 6. EPS yield and the synthesis of the key components of the EPS, including carbohydrate, protein, and lipid, were found maximum at pH 6. Changes in biofilm formation patterns and various topological parameters at varying pH/pCO2 conditions were observed. A cellular chaining pattern was observed at pH 4, and maximum biofilm formation was obtained at pH 6 with biomass of 5.28582 ± 0.5372 µm3/µm2 and thickness of 9.982 ± 1.5288 µm. Structural characterization of EPS showed changes in various functional groups of constituent macromolecules with varying pH. The amorphous nature of the EPS and the changes in linkages and associated functional groups (-R2CHOR, -CH3, and -CH2) with pH variation was confirmed. EPS showed a two-step degradation with a maximum weight loss of 59.147% and thermal stability up to 480 °C at pH 6. The present work efficiently demonstrates the role of EPS in providing structural and functional stability to the biofilm in varying pH conditions. The findings will provide a better understanding of the adaptability of marine bacteria in the future effect of ocean acidification.

Whole-genome sequencing of biofilm-forming and chromium-resistant mangrove fungus Aspergillus niger BSC-1.

Filamentous fungus Aspergillus niger has gained significant industrial and ecological value due to its great potential in enzymatic activities. The present study reports the complete genome sequence of A. niger BSC-1 which was isolated from Indian Sundarban mangrove ecosystem. The study revealed that the genome of A. niger BSC-1 was 35.1 Mbp assembled in 40 scaffolds with 49.2% GC content. A total of 10,709 genes were reported out of which 10,535 genes were predicted for encoding the proteins. BUSCO assessment showed 98.6% of genome completeness indicating high quality genome sequencing. The genome sequencing of A. niger BSC-1 revealed the presence of rodA and exgA genes for initial adhesion to surface and Ags genes for matrix formation, during biofilm growth. OrthoVenn2 analysis revealed that A.niger BSC-1 shared 9552 gene clusters with the reference strain A. niger CBS554.65. Semi-quantitative RT-PCR analysis unveiled the role of Ags1 and P-type ATPase in fungal biofilm formation and chromium (Cr) resistance, respectively. During biofilm growth the expression of Ags1 significantly (P < 0.0001; two-way ANOVA followed by Sidak's multiple comparisons test) increased with respect to planktonic culture revealing the possible involvement of Ags1 in biofilm matrix formation. Expression of P-type ATPase gene was significantly upregulated (P < 0.0001; one-way ANOVA followed by Dunnett's multiple comparisons test) with the increasing chromium concentration in the fungal culture. Besides, several other genes encoding metalloprotease, copper and zinc binding proteins, and NADH-dependent oxidoreductase were also found in the genome of A. niger BSC-1. These proteins are also involved in heavy metal tolerance and nanofabrication indicating that this filamentous fungus A. niger BSC-1 could be potentially utilized for chromium detoxification through biofilm or nanobiremediation.

Toll-interacting protein in the freshwater fish Labeo rohita exhibits conserved structural motifs of higher eukaryotes and is distinctly expressed in pathogen-associated molecular pattern stimulations and bacterial infections.

Toll-interacting protein (Tollip) is a critical regulator of TOLL- like receptor (TLR)-signaling pathway. It is predominantly associated with TLR2 and TLR4 during acute inflammatory conditions and inhibits the TLR-mediated nuclear factor-kappa activation by suppressing the autophosphorylation of interleukin-1 receptor-associated kinase and its kinase activity. This article describes the Tollip of Labeo rohita (LrTollip), a highly valuable freshwater fish from the Indian subcontinent. The full-length LrTollip complementary DNA (1412 nucleotides) encodes a 276-amino acid (aa) protein, depicting a highly conserved target of the Myb1 (Tom1)-binding domain (TBD; 1-53 aa), conserved core domain 2 (C2; 54-151 aa), and coupling of ubiquitin to endoplasmic reticulum degradation (CUE; 231-273 aa) domains of mouse and human counterparts. The key amino acids exerting the critical functions of Tollip, such as phospholipids recognition and ubiquitination, are present in the C2 and CUE domains of LrTollip, respectively. LrTollip is widely expressed in the kidneys, gills, spleen, liver, and blood, and among these tested tissues, the highest expression is observed in blood. In response to TLR ligands and NOD-like receptor (NLR) ligands stimulations and Aeromonas hydrophila, Edwardsiella tarda, and Bacillus subtilis infections, LrTollip gene expression is induced in various organs/tissues with remarkable difference in their kinetics. These data together suggest the important role of LrTollip in TLR- and NLR-signal transduction pathways and immune-related diseases in fish.

Apoptosis and autophagy modulating dietary phytochemicals in cancer therapeutics: Current evidences and future perspectives.

The global incidence of cancer and cancer-related mortality is expected to rise in recent years despite advancements in cancer diagnosis and therapeutics. Increasing evidences of decrypting molecular mechanisms underlying cancer progression have commanded the tremendous development of synthetic anticancer drugs. With limitations in the current conventional cancer therapeutic approaches, the non-nutritive dietary phytochemicals have emerged as potent modulators of apoptosis and autophagy associated key signaling pathways in various cancer cells. The dynamic regulation of apoptosis and autophagy by phytochemicals in cancer are identified as promising therapeutic candidates with minimal cytotoxicity and enhanced biological activity. Dietary phytochemicals and their synthetic analogs have exhibited potency in the modulation of apoptosis and autophagy in several cancer cells as individuals or in combination with pre-existing FDA (Food and Drug Administration) approved anticancer drugs. In the current generation of medical science, developing precision and personalized medicine and their consumption as food supplements will hold high prevalence in cancer therapeutics. Hence understating the impact of dietary phytochemicals on human health and their molecular mechanism will thrive a new horizon in cancer therapeutics. Hence, this review has emphasized the role of apoptotic/autophagy modulating dietary phytochemicals in cancer therapy, their preclinical and clinical applications and the future direction of enhanced nano-formulation for better clinical efficacy.

Useful autosomal STR marker sets for forensic and paternity applications in the Central Indian population.

BACKGROUND: Many countries have developed their core set of STR loci for forensic application and database generation, which India lacks. AIM: To assess the usefulness of various combinations of autosomal STR marker sets for their superior use in the central Indian population for forensic and paternity applications. SUBJECTS AND METHODS: 19 STR marker sets were analysed on 200 central Indian populations and 20 paternity cases to assess their usefulness. RESULTS: Two marker sets each comprising 19 STR markers are found to be superior to 20 expanded CODIS loci in the studied population. These marker sets also showed their effectiveness in 20 paternity cases having CPI values of 7.62 × 1011 and 7.16 × 1011. Three non-CODIS STR markers Penta E, Penta D, and SE33 showed amplification in 50 challenging samples with >0.80 heterozygosity. CONCLUSION: Population-specific STR marker sets are useful in forensic and paternity applications, as well as database generation, and it is envisioned that Penta E, Penta D, and SE33 markers will be included in the list of core STR loci in the central Indian population.

Prevalence and characterisation of size and sequence-based microvariant alleles at nine autosomal STR markers in the Central Indian population.

BACKGROUND: Though microvariant alleles are widely reported in global populations, they are not well characterised to date. AIM: To study the prevalence and characterisation of size and sequence-based microvariant alleles. SUBJECTS AND METHODS: Next Generation Sequencing (NGS) was used to sequence microvariant alleles at nine autosomal STR markers in 138 samples. RESULTS: After sequencing 31 STR markers using Precision ID GlobalFilerTM NGS STR panel v2, only nine markers, i.e. D12S391, D19S433, D1S1656, D21S11, D2S441, D7S820, FGA, Penta D, and TH01 showed the prevalence of microvariant alleles. Occurrence of microvariant alleles was positively correlated with Total Possible Alleles (p < 0.005), Power of Discrimination (p < 0.01), Polymorphic Information Content (p < 0.01), and Power of Exclusion (p < 0.05) and negatively correlated with the Matching Probability (p < 0.01). The average allele frequency of the microvariant alleles was found to be significantly less than the allele frequency value of the complete alleles (p = 0.88). Further, sequencing of these microvariant alleles reveals the deletion of nucleotides from the start, end, or middle of the repeat unit is responsible for the generation of a microvariant allele. CONCLUSIONS: Prevalence of microvariant alleles is rare in nature and is limited to 9 STR loci out of 31 STR loci tested in the central Indian population. The occurrence of microvariant alleles in a locus increases its forensic and paternity application.

Effect of superoxide dismutase, catalase, and glutathione reductase supplementation on cryopreservation of Black Bengal buck semen.

The present experiment was carried out with the objectives to study the effects of antioxidants superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GSH) on cryopreservation of Black Bengal buck semen. Semen ejaculates (n = 60) were collected from eight bucks by artificial vagina method and diluted with Tris citrate egg yolk glycerol extender. To study the effect of antioxidants, SOD was added @ 0, 100, and 150 IU/ml; CAT was added @ 0, 200, and 400 IU/ml while GSH was added @ 0, 1, and 2 mM of diluted semen. Semen samples were equilibrated and vapor frozen in liquid nitrogen. Semen samples were evaluated after 48 h of storage for post thaw in vitro characters such as motility, viability, functional membrane integrity, and acrosome integrity. Semen extenders supplemented with SOD @ 100 and 150 IU/ml and GSH @ 1 and 2 mM had a higher (p < 0.01) number of motile cells, viable cells, HOST reacted cells, and acrosome intact cells than their respective controls. Further, semen extenders added with catalase @ 200 and 400 IU/ml had more (p < 0.05) number of viable, HOST reacted cells and significantly higher (p < 0.01) acrosome intact sperm cells than its control group. It can be concluded that supplementation of antioxidants SOD, GSH, and CAT had a beneficial effect on cryopreservation of Black Bengal buck semen.

Molecular characterization and expressional modulation of IRAK1 as downstream signaling adaptor molecule of TLR-signaling pathways in Labeo rohita following PAMPs stimulation and bacterial infections.

Interleukin-1 receptor associated kinase (IRAK1) is one of the crucial signal transduction mediators in TLR/IL-1R signaling pathways in host immune system. To investigate about it in rohu (Labeo rohita), one of the economically important freshwater fish species in the Indian subcontinent, we cloned, characterized and analyzed its expression following bacterial infection and pathogens associated molecular patterns (PAMPs) stimulation. The full-length cDNA of rohu IRAK1 (LrIRAK1) consisted of 2765 nucleotide (nt) having an ORF of 2115 nt encoding a polypeptide of 704 amino acids (aa) with a molecular mass of 70.4 kDa. Structurally, LrIRAK1 consisted of twenty-nine helix, twelve strands and forty one coils making one N-terminal death domain (19-94 aa) and a central serine threonine kinase catalytic domain (or kinase domain) (188-489aa). In addition to these two prominent domains, LrIRAK1 also contained highly conserved amino acids viz., lysine 215 and aspartic acid 314 and threonine 185, 361 which were reported to be important for kinase and phosphorylation activity respectively in other animals. Similar to higher vertebrates, LrIRAK1 also consisted of CDK1 (cyclin-dependent kinase1) at 338-352 aa; NEK2 (NIMA-related kinase 2) at 47-61 aa; NEK6 (NIMA-related kinase 6) at 581-595 aa and AMPK (AMP- activated protein kinase) motif at 518-538 aa. Phylogenetically, LrIRAK1 is closely related to cave fish, common carp exhibiting high similarity (~95%) and identity (~90%). In the uninfected fish, the LrIRAK1 expression was highest in liver (~11.5 fold) and lowest in blood. In response to Aeromonas hydrophila, Edwardsiella tarda and Bacillus subtilis infection and various TLR and NLR-ligands stimulation, the expression of LrIRAK1 was markedly enhanced at various time points in almost all the tested tissues. These results together suggest the key role of LrIRAK1 in pattern recognition receptors (PRRs)-mediated host defense against pathogenic insults.

Alternatives to amelogenin markers for sex determination in humans and their forensic relevance.

Forensic DNA typing and subsequent molecular methods of sex determination in humans have been proven to be an imperious tool to criminal justice system. In current practice, most of the short tandem repeat (STR) based commercial kits contain amelogenin as the sexing marker. Amelogenin gene which contributes to the tooth enamel formation is present on both X and Y chromosome with a variation in base pair size. However, huge discrepancies have been observed with amelogenin based sex determination mostly due to X and Y deletion in the population and mutation in primer binding sites. Some ethnicities such as those in Indian population are affected badly with inappropriate sex determination by amelogenin marker due to the presence of high frequency of Y deletion in the population. Presence of PCR inhibitors, degradation in the DNA samples and presence of mixed DNA also contribute to the discrepancy in results obtained by amelogenin analysis. To overcome this problem, many alternative markers/techniques such as STS, SRY, TSPY, DXYS156, SNPs, DYZ1 and Next generation sequencing have been discussed in much detail with their respective pros and cons. In this regard, inclusion of one or more alternative markers along with amelogenin will decrease the anomalies in sex determination observed while using the amelogenin marker alone in forensic sample analysis.

Thanatomicrobiome and epinecrotic community signatures for estimation of post-mortem time interval in human cadaver.

Estimation of post-mortem time interval (PMI) is a key parameter in the forensic investigation which poses a huge challenge to the medico-legal experts. The succession of microbes within different parts of the human body after death has shown huge potential in the determination of PMI. Human body harbors trillions of microorganisms as commensals. With the death of an individual when biological functions are stopped, these microorganisms behave contrarily along with the invasion of degrading microbes from the environment. Human cadaver becomes a rich source of nutrients due to autolysis of cells, which attracts various invading microorganisms as well as macroorganisms. At different stages of degradation, the succession of microorganisms differs significantly which can be explored for accurate PMI estimation. With the advent of microbial genomics technique and reduction in the cost of DNA sequencing, thanatomicrobiome and epinecrotic community analysis have gained huge attention in PMI estimation. The article summarizes different sources of microorganisms in a human cadaver, their succession pattern, and analytical techniques for application in the field of microbial forensics. KEY POINTS: • Thanatomicrobiome and epinecrotic microbiome develop in postmortem human body. • Lack of metabolic, immune, neuroendocrine systems facilitate microbial succession. • Analysis of postmortem microbial communities predicts accurate PMI.

Biocompatible PHB Production from Bacillus Species Under Submerged and Solid-State Fermentation and Extraction Through Different Downstream Processing.

Catastrophic global accumulation of non-biodegradable plastic has led to efforts for production of alternative eco-friendly biopolymer. Here, we attempted to produce a biodegradable, cytocompatible and eco-friendly polyhydroxy-butyrate (PHB) from a pigmented Bacillus sp. C1 (2013) (KF626477) through submerged (SmF) and solid-state fermentation (SSF). Under SmF and SSF, 0.60 g l-1 and 1.56 g l-1 of PHB with 0.497 g l-1 of yellow fluorescent pigment (YFP) was produced. Fourier transform infrared (FTIR) absorption bands at 1719-1720 cm-1 indicate the presence of C=O group of PHB. Nuclear magnetic resonance (NMR) exhibited the typical chemical shift patterns of PHB, and crystallinity was confirmed from X-ray diffraction (XRD). The melting temperature (Tm), degradation temperature (Td) and crystallinity (Xc) of extracted PHB were found to be 171 °C, 288 °C and 35%, respectively. FACS (Fluorescence-activated cell sorting) confirmed cytocompatibility of PHB at 400 µg ml-1 in mouse fibroblast line. Moreover, biodegradability and elevated cytocompatibility of the PHB produced through SSF make them highly potential biomaterials to be used as a drug delivery carrier in future.