Exploring the Frozen Armory: Antiphage Defense Systems in Cold-Adapted Bacteria with a Focus on CRISPR-Cas Systems.

Our understanding of the antiphage defense system arsenal in bacteria is rapidly expanding, but little is known about its occurrence in cold-adapted bacteria. In this study, we aim to shed light on the prevalence and distribution of antiphage defense systems in cold-adapted bacteria, with a focus on CRISPR-Cas systems. Using bioinformatics tools, Prokaryotic Antiviral Defense LOCator (PADLOC) and CRISPRCasTyper, we mapped the presence and diversity of antiphage defense systems in 938 available genomes of cold-adapted bacteria from diverse habitats. We confirmed that CRISPR-Cas systems are less frequent in cold-adapted bacteria, compared to mesophilic and thermophilic species. In contrast, several antiphage defense systems, such as dXTPases and DRTs, appear to be more frequently compared to temperate bacteria. Additionally, our study provides Cas endonuclease candidates with a potential for further development into cold-active CRISPR-Cas genome editing tools. These candidates could have broad applications in research on cold-adapted organisms. Our study provides a first-time map of antiphage defense systems in cold-adapted bacteria and a detailed overview of CRISPR-Cas diversity.

Multi-Color-Emissive Magneto-Luminescent Nanoarchitectures for Targeted Identification of Heterogeneous Exosomes Associated with Lung Cancer Metastasis.

Due to the lack of early detection before metastasis and failure of current therapy to cure the disease, lung cancer contributes to the highest cancer-related mortality worldwide. Tenascin C (TNC) (+) exosomes promote metastasis, amphiregulin (AREG) (+) exosomes are associated with chemotherapy resistance, and programmed cell death ligand-1 (PDL-1) (+) exosomes are associated with immunotherapy resistance, and they are emerging as biomarkers in clinics. However, due to heterogeneity, rapid isolation and multiplex detection of these exosomes are challenging. Herein, we report the design of an antibody-conjugated multi-color (orange, yellow, and green)-emissive carbon dot (CD)-attached cobalt spinel ferrite (CoFe2O4)-based magneto-luminescent nanoarchitecture for targeted capturing and identification of TNC (+), AREG (+), and PDL-1(+) exosomes selectively and simultaneously from whole blood samples. More importantly, to capture and identify the targeted AREG (+) exosome from an infected whole-blood sample, an anti-AREG antibody-attached green (520 nm)-emissive CD-conjugated CoFe2O4 nanoparticle-based magnetic-green luminescence nanoarchitecture was developed. Similarly, an anti-PDL-1 antibody-attached orange (600 nm)-emissive CDs-based magnetic-orange luminescence nanoarchitecture has been produced to capture and identify the PDL-1 (+) exosome. Furthermore, an anti-TNC antibody-attached yellow (560 nm)-emissive CD-based magnetic-orange luminescent nanoarchitecture has been designed to capture and identify the TNC (+) exosome. Notably, our finding reveals that 100% TNC (+) exosomes can be captured and imaged selectively from an infected blood sample using an anti-TNC antibody-conjugated nanoarchitecture. In addition, 100% AREG (+) exosomes can be captured and imaged selectively using an anti-AREG antibody-conjugated nanoarchitecture. Moreover, 100% PDL-1 (+) exosomes can be captured and imaged selectively using an anti-PDL-1 antibody-conjugated nanoarchitecture. Furthermore, we have demonstrated that a multi-color-emissive nanoarchitecture can be used for capturing and imaging all three exosomes simultaneously.

Functional analysis of the Candida albicans ECE1 Promoter.

The formation of hyphae is a key virulence attribute of Candida albicans as they are required for adhesion to and invasion of host cells, and ultimately deep-tissue dissemination. Hyphae also secrete the peptide toxin candidalysin, which is crucial for destruction of host cell membranes. The peptide is derived from a precursor protein encoded by the gene ECE1 which is strongly induced during hyphal growth. Previous studies revealed a very complex regulation of this gene involving several transcription factors. However, the promoter of the gene is still not characterized. Here, we present a functional analysis of the intergenic region upstream of the ECE1 gene. Rapid amplification of cDNA ends (RACE)-PCR was performed to identify the 5' untranslated region, which has a size of 49 bp regardless of the hyphae-inducing condition. By using green fluorescent protein (GFP) reporter constructs we further defined a minimal promoter length of 1,500 bp which was verified by RT-qPCR. Finally, we identified the TATA element required for the expression of the gene. It is located 106 to 109 bp upstream of the ECE1 start codon. Our results illustrate that despite a very short 5' UTR, a relatively long promoter is required to secure ECE1 transcription, indicating a complex regulatory machinery tightly controlling the expression of the gene. IMPORTANCE In recent years it was shown that secretion of the toxic peptide candidalysin from hyphae of the major human fungal pathogen Candida albicans contributes heavily to its virulence. The peptide is derived from a precursor protein which is encoded by the ECE1 gene whose transcription is known to be closely associated with formation of hyphae. Here, we used a GFP reporter system to determine the length of the ECE1 promoter and were able to show that it has a minimal size of 1,500 bp. Surprisingly, the gene has a very short 5' UTR of only 49 bp. In accordance with this, the TATA element required for transcription is located 106 to 109 bp upstream of the start codon. This indicates that ECE1 expression is controlled by a very long promoter allowing a complex network of transcription factors to contribute to the gene's regulation.

Comparative analysis of HiSeq3000 and BGISEQ-500 sequencing platform over whole genome sequencing metagenomics data.

Recent advances in sequencing technologies and platforms have enabled to generate metagenomics sequences using different sequencing platforms. In this study, we analyzed and compared shotgun metagenomic sequences generated by HiSeq3000 and BGISEQ-500 platforms from 12 sediment samples collected across the Norwegian coast. Metagenomics DNA sequences were normalized to an equal number of bases for both platforms and further evaluated by using different taxonomic classifiers, reference databases, and assemblers. Normalized BGISEQ-500 sequences retained more reads and base counts after preprocessing, while a slightly higher fraction of HiSeq3000 sequences were taxonomically classified. Kaiju classified a higher percentage of reads relative to Kraken2 for both platforms, and comparison of reference database for taxonomic classification showed that MAR database outperformed RefSeq. Assembly using MEGAHIT produced longer assemblies and higher total contigs count in majority of HiSeq3000 samples than using metaSPAdes, but the assembly statistics notably improved with unprocessed or normalized reads. Our results indicate that both platforms perform comparably in terms of the percentage of taxonomically classified reads and assembled contig statistics for metagenomics samples. This study provides valuable insights for researchers in selecting an appropriate sequencing platform and bioinformatics pipeline for their metagenomics studies.

Whole-Genome-Based Web Genomic Resource for Water Buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis), belonging to the Bovidae family, is an economically important animal as it is the major source of milk, meat, and drought in numerous countries. It is mainly distributed in tropical and subtropical regions with a global population of approximately 202 million. The advent of low cost and rapid sequencing technologies has opened a new vista for global buffalo researchers. In this study, we utilized the genomic data of five commercially important buffalo breeds, distributed globally, namely, Mediterranean, Egyptian, Bangladesh, Jaffrarabadi, and Murrah. Since there is no whole-genome sequence analysis of these five distinct buffalo breeds, which represent a highly diverse ecosystem, we made an attempt for the same. We report the first comprehensive, holistic, and user-friendly web genomic resource of buffalo (BuffGR) accessible at http://backlin.cabgrid.res.in/buffgr/, that catalogues 6028881 SNPs and 613403 InDels extracted from a set of 31 buffalo tissues. We found a total of 7727122 SNPs and 634124 InDels distributed in four breeds of buffalo (Murrah, Bangladesh, Jaffarabadi, and Egyptian) with reference to the Mediterranean breed. It also houses 4504691 SSR markers from all the breeds along with 1458 unique circRNAs, 37712 lncRNAs, and 938 miRNAs. This comprehensive web resource can be widely used by buffalo researchers across the globe for use of markers in marker trait association, genetic diversity among the different breeds of buffalo, use of ncRNAs as regulatory molecules, post-transcriptional regulations, and role in various diseases/stresses. These SNPs and InDelscan also be used as biomarkers to address adulteration and traceability. This resource can also be useful in buffalo improvement programs and disease/breed management.

Blocking SARS-CoV-2 Delta Variant (B.1.617.2) Spike Protein Receptor-Binding Domain Binding with the ACE2 Receptor of the Host Cell and Inhibiting Virus Infections Using Human Host Defense Peptide-Conjugated Graphene Quantum Dots.

The emergence of double mutation delta (B.1.617.2) variants has dropped vaccine effectiveness against SARS-CoV-2 infection. Although COVID-19 is responsible for more than 5.4 M deaths till now, more than 40% of infected individuals are asymptomatic carriers as the immune system of the human body can control the SARS-CoV-2 infection. Herein, we report for the first time that human host defense neutrophil α-defensin HNP1 and human cathelicidin LL-37 peptide-conjugated graphene quantum dots (GQDs) have the capability to prevent the delta variant virus entry into the host cells via blocking SARS-CoV-2 delta variant (B.1.617.2) spike protein receptor-binding domain (RBD) binding with host cells' angiotensin converting enzyme 2 (ACE2). Experimental data shows that due to the binding between the delta variant spike protein RBD and bioconjugate GQDs, in the presence of the delta variant spike protein, the fluorescence signal from GQDs quenched abruptly. Experimental quenching data shows a nonlinear Stern-Volmer quenching profile, which indicates multiple binding sites. Using the modified Hill equation, we have determined n = 2.6 and the effective binding affinity 9 nM, which is comparable with the ACE2-spike protein binding affinity (8 nM). Using the alpha, beta, and gamma variant spike-RBD, experimental data shows that the binding affinity for the delta B.1.617.2 variant is higher than those for the other variants. Further investigation using the HEK293T-human ACE2 cell line indicates that peptide-conjugated GQDs have the capability for completely inhibiting the entry of delta variant SARS-CoV-2 pseudovirions into host cells via blocking the ACE2-spike protein binding. Experimental data shows that the inhibition efficiency for LL-37 peptide- and HNP1 peptide-attached GQDs are much higher than that of only one type of peptide-attached GQDs.

Genome-Wide Association Studies in Diverse Spring Wheat Panel for Stripe, Stem, and Leaf Rust Resistance.

Among several important wheat foliar diseases, Stripe rust (YR), Leaf rust (LR), and Stem rust (SR) have always been an issue of concern to the farmers and wheat breeders. Evolution of virulent pathotypes of these rusts has posed frequent threats to an epidemic. Pyramiding rust-resistant genes are the most economical and environment-friendly approach in postponing this inevitable threat. To achieve durable long term resistance against the three rusts, an attempt in this study was made searching for novel sources of resistant alleles in a panel of 483 spring wheat genotypes. This is a unique and comprehensive study where evaluation of a diverse panel comprising wheat germplasm from various categories and adapted to different wheat agro-climatic zones was challenged with 18 pathotypes of the three rusts with simultaneous screening in field conditions. The panel was genotyped using 35K SNP array and evaluated for each rust at two locations for two consecutive crop seasons. High heritability estimates of disease response were observed between environments for each rust type. A significant effect of population structure in the panel was visible in the disease response. Using a compressed mixed linear model approach, 25 genomic regions were found associated with resistance for at least two rusts. Out of these, seven were associated with all the three rusts on chromosome groups 1 and 6 along with 2B. For resistance against YR, LR, and SR, there were 16, 18, and 27 QTL (quantitative trait loci) identified respectively, associated at least in two out of four environments. Several of these regions got annotated with resistance associated genes viz. NB-LRR, E3-ubiquitin protein ligase, ABC transporter protein, etc. Alien introgressed (on 1B and 3D) and pleiotropic (on 7D) resistance genes were captured in seedling and adult plant disease responses, respectively. The present study demonstrates the use of genome-wide association for identification of a large number of favorable alleles for leaf, stripe, and stem rust resistance for broadening the genetic base. Quick conversion of these QTL into user-friendly markers will accelerate the deployment of these resistance loci in wheat breeding programs.

Improved chemotherapy against breast cancer through immunotherapeutic activity of fucoidan decorated electrostatically assembled nanoparticles bearing doxorubicin.

Immunotherapeutic nanoparticles (NPs) could be a viable option for delivering cytotoxic agents in a manner which suppresses their toxic manifestations. Doxorubicin (DOX) loaded NPs were prepared using fucoidan (FCD), an immunomodulatory polysaccharide and evaluated against cancer. FCD was electrostatically assembled with cationic polyethylenimine (PEI) through intermolecular electrostatic interactions to develop an immunomodulatory platform to deliver DOX. FCD NPs offered improved cytotoxicity (2.64 folds), cell cycle arrest in G1-S phase (34.65%) and apoptosis (66.12%) in tumor cells compared to free DOX. The enhanced apoptosis was due to raised mitochondrial depolarization (88.00%). In vivo anticancer activity in 4T1 induced tumor bearing BALB/c mice demonstrated a 2.95 folds enhanced efficacy of NPs. Importantly, NPs treatment generated an immunotherapeutic response indicated by gradual increment of the plasma IL-12 levels and reversed polarization of tumor associated macrophages (TAMs) towards M1 subtype. Furthermore, pharmacokinetic study suggested that NPs administration in tumor infested mice caused serum DOX levels to vary in a biphasic pattern, with twin peaks occurring at 1 h and 6 h which help in maintaining preferential drug localization in tumor. Developed NPs would be an excellent approach for improved immune-chemotherapy (in terms of efficacy, safety and immunocompetency) against cancer.

Retraction Note to: Environmental impact of plantations in and around the petroleum refinery: a case study.

This article [1] has been retracted at the request of the Editor-in-Chief. Significant sections of the article show a similarity with the author's own article published previously [2]. All the authors agree with the retraction.

Nebulized N-Acetylcysteine for Management of Plastic Bronchitis.

BACKGROUND: Plastic bronchitis is characterized by formation of extensive obstructive endobronchial casts and high recurrence rates. CASE CHARACTERISTICS: Two children (1-year-old girl, 7-year-old boy) who had recurrent episodes of respiratory distress with acute worsening. Bronchoscopy revealed membrane-like casts. Both children were managed with nebulized N-acetylcysteine in addition to management for asthma. OUTCOME: Symptom-free without recurrence for more than 9 months of follow-up. MESSAGE: Nebulized N- acetylcysteine may be helpful in prevention of recurrence of plastic bronchitis due to asthma.

Carbomer gel bearing methotrexate loaded lipid nanocontainers shows improved topical delivery intended for effective management of psoriasis.

The present investigation reports the evaluation of potential use of Carbomer gel bearing methotrexate loaded nanostructured lipid carriers for topical application of methotrexate for possible therapy of psoriasis in comparison to solid lipid nanoparticles. These were evaluated for various parameters such as particle size, surface charge, entrapment efficiency, shape and surface morphology, thermal analysis, in-vitro drug release through skin (Franz diffusion cell) and drug deposition study, fluorescence microscopy, particle-skin interaction study, skin-irritation testing and storage stability. The formulation (NLC5) showed the best entrapment efficiency (62.72 ±â€¯0.94%) while SLN showed only 26.84 ±â€¯0.64% with particle size of 221 ±â€¯14nm and 212 ±â€¯11nm, respectively. Skin permeation study of MTX loaded SLN and NLC5 hydrogels showed prolonged drug release up to 24 h. The skin drug deposition study showed the greatest deposition of drug enriched NLC5 hydrogel (28.8%) when compared to plain drug enriched hydrogel (11.4%) and drug enriched SLN hydrogel (18.6%). Fluorescence microscopy suggested the localization effect of these lipid based systems to deeper skin region. The primary skin irritation studies indicated that MTX loaded SLN or NLC5 hydrogels resulted no erythema. It can be concluded that NLC represents a promising particulate carrier having prolonged drug release, improved skin permeation.

Fabrication of 3-O-sn-Phosphatidyl-L-serine Anchored PLGA Nanoparticle Bearing Amphotericin B for Macrophage Targeting.

PURPOSE: To fabricate, characterize and evaluate 3-O-sn-Phosphatidyl-L-serine (PhoS) anchored PLGA nanoparticles for macrophage targeted therapeutic intervention of VL. MATERIALS AND METHODS: PLGA-AmpB NPs were prepared by well-established nanoprecipitation method and decorated with Phos by thin film hydration method. Physico-chemical characterization of the formulation was done by Zetasizer nano ZS and atomic force microscopy. RESULTS: The optimized formulation (particle size, 157.3 ± 4.64 nm; zeta potential, - 42.51 ± 2.11 mV; encapsulation efficiency, ∼98%) showed initial rapid release up to 8 h followed by sustained release until 72 h. PhoS generated 'eat-me' signal driven augmented macrophage uptake, significant increase in in-vitro (with ∼82% parasite inhibition) and in-vivo antileishmanial activity with preferential accumulation in macrophage rich organs liver and spleen were found. Excellent hemo-compatibility justified safety profile of developed formulation in comparison to commercial formulations. CONCLUSION: The developed PhoS-PLGA-AmpB NPs have improved efficacy, and necessary stability which promisingly put itself as a better alternative to available commercial formulations for optimized treatment of VL.

Prediction of miRNA and Identification of their Relationship Network Related to Late Blight Disease of Potato.

BACKGROUND: Late blight is a serious disease in potato caused by Phytophthora infestans. To date only few miRNA have been discovered which are related to late blight disease of potato during host pathogen interaction. Recent studies showed that miRNA, an important gene expression regulator, plays a very important role in host-pathogen interaction by silencing genes either by destructing or blocking of translation of mRNA. METHOD: Homology search was performed between non-redundant mature miRNA sequences from miRBase database and Solanum tuberosum EST sequences from NCBI database. Screening of the potential miRNA was done after secondary structure prediction. The target related to late blight disease of respective miRNA was functionally annotated. To identify the relationship between the predicted and mature miRNAs, multiple sequence alignment and evolutionary relationships were established. RESULTS AND CONCLUSION: 34 Candidate miRNA related to late blight disease of potato were identified which were associated to five target genes. These miRNAs were linked with Avr3a, INF1, INF2b genes which are elicitin like protein and triggers a hypersensitive response to host cell. Mapping of target sequences showed similarity with Solanum lycopersicum NRC1 gene of chr.1, which are reported as a casual protein required for Pto-mediated cell death and resistance in N. benthamiana. NRC1 are considered as a RX-CC_like domain-containing protein which shows similarity with coiledcoil domain of the potato virus X resistance protein (RX) in Solanum tuberosum. RX recognizes pathogen effector proteins and triggers a response that may be as severe as localized cell death thereby providing resistance against potato virus X.

Enhanced apoptosis, survivin down-regulation and assisted immunochemotherapy by curcumin loaded amphiphilic mixed micelles for subjugating endometrial cancer.

Survivin is up-regulated in 83% of endometrial cancer leading to resistance development. As endometrial tumor advances, it also elicits chronic inflammation characterized by increased cytokine secretion and immune cells infiltration. The present study was designed to engineer mixed micellar curcumin loaded formulation for investigating survivin down-regulation, its anti-cancer and cytokine modulatory potential against endometrial cancer Ishikawa cells. Flory-Huggins interaction parameter (χpd) was applied to predict the compatibility between curcumin and surfactant mixture. The developed and characterized formulations were used to comparatively assess hemolysis, cellular uptake, cell-viability, apoptosis, mitochondrial membrane potential loss, rhodamine accumulation and bioavailability. In-vitro cytotoxicity in Vero cells demonstrated no deleterious effects on cell population. We saw better bioavailability, significant rhodamine accumulation, changes in protein expression and modulation in TNF-α, IL-6 and IL-10 levels. In conclusion, developed formulation warrants exploring the therapeutic interventions for overcoming resistance development in endometrial cancer.

Targeting tumor associated macrophages (TAMs) via nanocarriers.

Recruitment of inflammatory cells to tumor has been well documented, with the most frequent inhabitants being macrophages termed as tumor associated macrophages, (TAMs). Their presence was thought to be an evidence of immune system initiating a fight response towards the tumor, i.e. immune surveillance. This is the case too initially, when TAMs majorly exhibit an M1 phenotype, but their continued presence in tumor microenvironment brings about their polarization to M2 phenotype, which not only participate in continued sustenance of existing tumor but also open up deleterious avenues for further progression and metastasis of cancer. Current perspective is built around this very premise and focuses specifically on TAMs and how they are being targeted by researchers working in annals of nanomedicine. To do so, we dwell into tumor microenvironment and focus on nanotechnology based drug delivery aspects which have either been already or can be potentially employed in the future to target tumor associated macrophages for improved immunoadjuvant therapy of cancer.

Lessons Learned from Gemcitabine: Impact of Therapeutic Carrier Systems and Gemcitabine's Drug Conjugates on Cancer Therapy.

Currently, drug delivery systems have a high impact in cancer therapy and are receiving more attention than conventional cancer treatment modalities. Compared with current cancer therapies, gemcitabine (2', 2'-difluoro-2'-deoxycytidine) has been proven to be an effective chemotherapeutic agent against pancreatic, colon, bladder, breast, ovarian, non-small-cell lung, and head and neck cancers in combination with other anticancer agents. To improve the safety and efficacy of cytotoxic drugs, several drug delivery systems have been explored. This review outlines the recent work directed toward gemcitabine delivery systems for cancer therapy, including aerosols, polymeric nanoparticles, liposomes, microparticles, carbon nanotubes, and multifunctional theranostic nanomedicines. It also provides insight into the design and development of gemcitabine conjugation for safe and effective cancer therapy. Despite the clinical promises of gemcitabine, many therapeutic challenges remain. Specifically, its therapeutic use in cancer chemotherapy is impeded by a short biological half-life, caused by its rapid metabolism, and resistance due to increased expression of ribonucleotide reductase. In our opinion, many research investigations have contributed to improve the selectivity and efficacy of gemcitabine. This combined approach of drug delivery systems and gemcitabine conjugates has shown promising efficacy in preclinical models and significant potential for future clinical cancer-therapeutic applications. Also, these strategies overcome most of the aforementioned limits of gemcitabine.

Prevalence, Response to Cysticidal Therapy, and Risk Factors for Persistent Seizure in Indian Children with Neurocysticercosis.

Background. Neurocysticercosis (NCC) is the commonest cause of childhood acquired epilepsy in developing countries. The use of cysticidal therapy in NCC, except "single lesion NCC," is still debated in view of its doubtful usefulness and potential adverse effects. Methods. Children presenting with first episode of seizure or acute focal neurological deficit without fever were screened for NCC and received appropriate therapy (followup done for 1 year to look for the response and side effects). Results. The prevalence of NCC was 4.5%. Most common presenting feature was generalized seizure and commonest imaging finding was single small enhancing lesion in the parietal lobe. Abnormal EEG and CSF abnormalities were found in almost half of the children. The response to therapy was very good with infrequent recurrence of seizure and adverse effects of therapy were encountered rarely. No risk factors for persistent seizure could be identified. Conclusion. Present study shows that the response to cysticidal therapy is very good in NCC as seizure recurrence was observed in only 5%, 4.2%, and 4.2% of cases at 3-month, 6-month, and 1-year followup. Adverse effects of therapy were observed in 20% of cases during therapy but they were mild and self-limiting.

Nanosized complexation assemblies housed inside reverse micelles churn out monocytic delivery cores for bendamustine hydrochloride.

OBJECTIVE: We explore a plausible method of targeting bendamustine hydrochloride (BM) to circulatory monocytes by exploiting their intrinsic endocytic/phagocytic capability. METHODS: We do so by complexation of sodium alginate and chitosan inside dioctyl sulfo succinate sodium (AOT) reverse micelles to form bendamustine hydrochloride loaded nanoparticles (CANPs). Dynamic light scattering, electrophoretic mobility and UV spectroscopy were used to detail intra-micellar complexation dynamics and to prove that drug was co-captured during interaction of carbohydrate polymers. A fluorescent conjugate of drug (RBM) was used to trace its intracellular fate after its loading into nanoparticles. RESULTS: CANPs were sized below 150nm, had 75% drug entrapment and negative zeta potential (-30mV). Confocal microscopy demonstrated that developed chitosan alginate nanoparticles had the unique capability to carry BM specifically to its site of action. Quantitative and mechanism based cell uptake studies revealed that monocytes had voracious capacity to internalize CANPs via simultaneous scavenger receptor based endocytic and phagocytic mechanism. Comparative in vitro pharmacokinetic studies revealed obtainment of significantly greater intracellular drug levels when cells were treated with CANPs. This caused reduction in IC50 (22.5±2.1µg/mL), enhancement in G2M cell cycle arrest, greater intracellular reactive oxygen species generation, and increased apopotic potential of bendamustine hydrochloride in THP-1 cells. CONCLUSION: Selective monocytic targeting of bendamustine hydrochloride using carbohydrate constructs can prove advantageous in case of leukemic disorders displaying overabundance of such cells.