Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

HU (Histone‐like protein from Escherichia coli strain U93) is the most conserved nucleoid‐associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single‐molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface‐exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over‐condensed and mis‐segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid‐associated naRNAs, each previously implicated in HU’s high‐affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure‐binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.     

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs.To determine the variants of SARS-CoV-2 circulating in the state of Nevada,specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform,which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads,without the need of culture-based amplification.High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak.We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine(F) from proline (P),in the first reported isolate of SARS-CoV-2,Wuhan-Hu-1.This 323F variant was present at a very high frequency in Northern Nevada.Structural modeling determined this mutation in the interface domain,which is important for the association of accessory proteins required for the polymerase.In conclusion,we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations,which is important for understanding the evolution and circulation of SARS-CoV-2variants of public health importance,while it circulates in humans.     

MAPK signaling regulates c‐MYC for melanoma cell adaptation to asparagine restriction

Amino acid restriction is among promising potential cancer treatment strategies. However, cancer cells employ a multitude of mechanisms to mount resistance to amino acid restriction, which impede the latter’s clinical development. Here we show that MAPK signaling activation in asparagine‐restricted melanoma cells impairs GSK3‐β‐mediated c‐MYC degradation. In turn, elevated c‐MYC supports ATF4 translational induction by enhancing the expression of the amino acid transporter SLC7A5, increasing the uptake of essential amino acids, and the subsequent maintenance of mTORC1 activity in asparagine‐restricted melanoma cells. Blocking the MAPK‐c‐MYC‐SLC7A5 signaling axis cooperates with asparagine restriction to effectively suppress melanoma cell proliferation. This work reveals a previously unknown axis of cancer cell adaptation to asparagine restriction and informs mechanisms that may be targeted for enhanced therapeutic efficacy of asparagine limiting strategies.     

Role of extremophiles and their extremozymes in biorefinery process of lignocellulose degradation

Extremophiles - Technological advances in the field of life sciences have led to discovery of organisms that live in harsh environmental conditions referred to as extremophiles. These organisms...     

Inactivation of ntcA gene revealed differential proteome expression and induction of some hypothetical proteins in the cyanobacterium Anabaena sp. PCC 7120 and its derivative ntcA mutant under different levels of calcium

Abstract

Calcium is an important macronutrient for both prokaryotes and eukaryotes. It acts as an important second messenger mediating rapid response to environmental conditions. The present investigation deals with proteome profiling of Anabaena 7120 and its derivative ntcA mutant in response to varied calcium doses (0, 1 and 10?mM CaCl₂). Concentration of 1?mM CaCl₂ salt was the optimum concentration whereas 10?mM CaCl₂ was the inhibitory concentration for both the wild type and mutant strains. The results showed highly significant alteration in terms of protein abundance and differential response related to key processes of photosynthesis, energy and metabolism, nitrogen metabolism, oxidative and antioxidative defence, transport and signalling and fatty acid metabolism. In the wild type proteins related to photosynthesis and nitrogen metabolism showed upregulation at 1?mM CaCl₂ concentration while antioxidative defence related proteins were down-regulated. In the mutant however, proteins related to photosynthesis and nitrogen metabolism exhibited severe down-regulation. Some hypothetical proteins were also realized during proteome analysis. Overall, our results suggested that NtcA have a potential role in regulation of calcium ion dependent key processes underlying in various metabolic activities of the cyanobacterium Anabaena 7120.     

Magnetic core-shell nanoparticles for drug delivery by nebulization

Background

Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe₃O₄ magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated.

Results

Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting.

Conclusion

We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.     

A hydrolytic ��-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

??-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with ??-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni²⁺-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t _1/2 of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Pt??-GT shared 37 % sequence identity and 53 % homology with an extremophile ??-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.