Nanotechnology-based theranostic and prophylactic approaches against SARS-CoV-2.

SARS-CoV-2 (COVID-19) pandemic has been an unpredicted burden on global healthcare system by infecting over 700 million individuals, with approximately 6 million deaths worldwide. COVID-19 significantly impacted all sectors, but it very adversely affected the healthcare system. These effects were much more evident in the resource limited part of the world. Individuals with acute conditions were also severely impacted. Although classical COVID-19 diagnostics such as RT-PCR and rapid antibody testing have played a crucial role in reducing the spread of infection, these diagnostic techniques are associated with certain limitations. For instance, drawback of RT-PCR diagnostics is that due to degradation of viral RNA during shipping, it can give false negative results. Also, rapid antibody testing majorly depends on the phase of infection and cannot be performed on immune compromised individuals. These limitations in current diagnostic tools require the development of nanodiagnostic tools for early detection of COVID-19 infection. Therefore, the SARS-CoV-2 outbreak has necessitated the development of specific, responsive, accurate, rapid, low-cost, and simple-to-use diagnostic tools at point of care. In recent years, early detection has been a challenge for several health diseases that require prompt attention and treatment. Disease identification at an early stage, increased imaging of inner health issues, and ease of diagnostic processes have all been established using a new discipline of laboratory medicine called nanodiagnostics, even before symptoms have appeared. Nanodiagnostics refers to the application of nanoparticles (material with size equal to or less than 100 nm) for medical diagnostic purposes. The special property of nanomaterials compared to their macroscopic counterparts is a lesser signal loss and an enhanced electromagnetic field. Nanosize of the detection material also enhances its sensitivity and increases the signal to noise ratio. Microchips, nanorobots, biosensors, nanoidentification of single-celled structures, and microelectromechanical systems are some of the most modern nanodiagnostics technologies now in development. Here, we have highlighted the important roles of nanotechnology in healthcare sector, with a detailed focus on the management of the COVID-19 pandemic. We outline the different types of nanotechnology-based diagnostic devices for SARS-CoV-2 and the possible applications of nanomaterials in COVID-19 treatment. We also discuss the utility of nanomaterials in formulating preventive strategies against SARS-CoV-2 including their use in manufacture of protective equipment, formulation of vaccines, and strategies for directly hindering viral infection. We further discuss the factors hindering the large-scale accessibility of nanotechnology-based healthcare applications and suggestions for overcoming them.

CaMKK2 and CHK1 phosphorylate human STN1 in response to replication stress to protect stalled forks from aberrant resection.

Keeping replication fork stable is essential for safeguarding genome integrity; hence, its protection is highly regulated. The CTC1-STN1-TEN1 (CST) complex protects stalled forks from aberrant MRE11-mediated nascent strand DNA degradation (NSD). However, the activation mechanism for CST at forks is unknown. Here, we report that STN1 is phosphorylated in its intrinsic disordered region. Loss of STN1 phosphorylation reduces the replication stress-induced STN1 localization to stalled forks, elevates NSD, increases MRE11 access to stalled forks, and decreases RAD51 localization at forks, leading to increased genome instability under perturbed DNA replication condition. STN1 is phosphorylated by both the ATR-CHK1 and the calcium-sensing kinase CaMKK2 in response to hydroxyurea/aphidicolin treatment or elevated cytosolic calcium concentration. Cancer-associated STN1 variants impair STN1 phosphorylation, conferring inability of fork protection. Collectively, our study uncovers that CaMKK2 and ATR-CHK1 target STN1 to enable its fork protective function, and suggests an important role of STN1 phosphorylation in cancer development.

Modified Impedance Sensing System Determination of Virulence Characteristics of Pathogenic Bacteria Klebsiella Species.

An impedance sensing system is a family of biosensors that measure changes in electrical impedance to perform their functions. Physical and chemical changes in the impedance of the sensing element, such as changes in the concentration of a target analyte or changes in the physical properties of the sensing element, can result in changes in the impedance of the sensing element. Many impedance biosensors have been developed for the detection of pathogens in the past few decades. Several types of biosensors have been developed for the detection of infections, including transduction elements, biorecognition components, and electrochemical approaches. In this review, we discuss the characteristics and pathogenic factors associated with 2,3-butanediol-producing Klebsiella pneumoniae collected using impedance sensors. An impedance sensing system was introduced as a great method for monitoring the virulence factors of Klebsiella spp. in situ. Klebsiella pneumoniae produces virulence factors, including capsules, lipopolysaccharides, fimbriae, and siderophores, as part of its pathogenesis. It is possible to examine virulence factors' pathogenic characteristics in vitro and in vivo using real tissues or mouse models in order to conduct experiments. For the monitoring of virulence factors in situ, a novel alternative method has been developed to mimic the environment of real tissues. For the purpose of developing tissue-mimicking models, mucin and mannose were used to modify the surface of gold electrodes. These components are known to contribute to the adhesion of pathogens to epithelial cells in mammals.

Deficiency in mammalian STN1 promotes colon cancer development via inhibiting DNA repair.

Despite the high lethality of colorectal cancers (CRCs), only a limited number of genetic risk factors are identified. The mammalian ssDNA-binding protein complex CTC1-STN1-TEN1 protects genome stability, yet its role in tumorigenesis is unknown. Here, we show that attenuated CTC1/STN1 expression is common in CRCs. We generated an inducible STN1 knockout mouse model and found that STN1 deficiency in young adult mice increased CRC incidence, tumor size, and tumor load. CRC tumors exhibited enhanced proliferation, reduced apoptosis, and elevated DNA damage and replication stress. We found that STN1 deficiency down-regulated multiple DNA glycosylases, resulting in defective base excision repair (BER) and accumulation of oxidative damage. Collectively, this study identifies STN1 deficiency as a risk factor for CRC and implicates the previously unknown STN1-BER axis in protecting colon tissues from oxidative damage, therefore providing insights into the CRC tumor-suppressing mechanism.

Children's SARS-CoV-2 Infection and Their Vaccination.

SARS-CoV-2, a novel coronavirus, causes respiratory tract infections and other complications in affected individuals, and has resulted in numerous deaths worldwide. The unprecedented pace of its transmission worldwide, and the resultant heavy burden on healthcare systems everywhere, prompted efforts to have effective therapeutic strategies and vaccination candidates available to the global population. While aged and immunocompromised individuals form a high-risk group for COVID-19 and have severe disease outcome, the rate of infections among children has also increased with the emergence of the Omicron variant. In addition, recent reports of threatening SARS-CoV-2-associated complications in children have brought to the forefront an urgent necessity for vaccination. In this article, we discuss the current scenario of SARS-CoV-2 infections in children with a special focus on the differences in their immune system response as compared to adults. Further, we describe the various available COVID-19 vaccines, including the recent bivalent vaccines for children, in detail, intending to increase willingness for their acceptance.

Neutralizing antibody: a savior in the Covid-19 disease.

Coronavirus outbreak was declared a pandemic by World Health Organization (WHO) in March 2020. The pandemic has led to a devastating loss of life. It has shown us how infectious diseases can cause human existence at stake, and community health is important. The spike protein is the most immunogenic component of the virus. Most vaccine development strategies have focused on the receptor-binding domain (RBD) in the spike protein because it is the most specific target site that recognizes and interacts with human lung cells. Neutralizing antibodies are generated by the humoral immune system and reduce the viral load by binding to spike protein components. Neutralizing antibodies are the proteins secreted by plasma cells and serve as an important part of the defense mechanism. In the recent Covid-19 infection, neutralizing antibodies can be utilized for both diagnostic such as immune surveillance and therapeutic tools such as plasma therapy. So far, many monoclonal antibodies are in the clinical trial phase, and few of them are already in use. In this review, we have discussed details about neutralizing antibodies and their role in combating Covid-19 disease.

PARP-1 induces EMT in non-small cell lung carcinoma cells via modulating the transcription factors Smad4, p65 and ZEB1.

AIM: To study the role of PARP-1 in EMT of non-small cell lung carcinoma. MATERIALS AND METHODS: We used H1299 and H460 lung cancer cells for knockdown study of PARP-1 using shPARP-1 lentiviral particle. We performed western blotting, confocal microscopy, semi-quantitative PCR, wound healing and colony formation assays. BACKGROUND AND KEY FINDINGS: PARP-1 (poly-ADP ribose polymerase-1) is a multi-domain protein having DNA binding, auto-modification and catalytic domain, that participates in many biological processes including DNA damage detection and repair, transcription regulation, apoptosis, necrosis, cancer progression and metastasis. Metastasis is a leading cause of death in cancer patients, which starts in epithelial tumors via initiating epithelial to mesenchymal transition. There are various transcription factors involved in EMT including Snail-1, Smads, p65, ZEB1 and Twist1. We studied the effect of PARP-1 knockdown on EMT in non-small cell lung cancer cell line H1299. We found a significant increase in epithelial marker including ZO1 and ß-catenin, while prominent decrease in the mesenchymal marker vimentin after PARP-1 knockdown in H1299 cells. Transcription factors including p65, Smad4 and ZEB1 showed significant decrease with concurrent expression of EMT markers. Cell migration and colony formation decreased after PARP-1 knockdown in H1299 cells. SIGNIFICANCE: Overall, the shRNA mediated knockdown of PARP-1 in H1299 cells resulted in reversal of EMT or mesenchymal to epithelial transition (MET) characterized by an increase in epithelial markers and a decrease in mesenchymal markers, via down-regulating transcription factors including Smad4, p65 and ZEB1. Thus PARP-1 has a role in EMT in lung cancer.

An assessment of poly (ADP-ribose) polymerase-1 role in normal and cancer cells.

Poly (ADP-ribose) polymerase (PARP) is a superfamily of 18 proteins characterized by the PARP homology domain, the catalytic domain. This catalytic domain helps in the ADP-ribosylation of various acceptor proteins using nicotinamide adenine dinucleotide (NAD+) as a donor for ADP-ribose. PARP-1 and PARP-2 carry out 80% of poly-ADP-ribosylation of cellular protein. Hence, their combined knockout results in embryonic lethality of mice. PARP-1 consists of three major domains, namely, DNA binding domain, automodification domain, and a catalytic domain. These domains further consist of subdomains and motifs, which helps PARP-1 in a diverse function. PARP-1 is mainly involved in DNA damage detection and repair, but emerging evidence suggests its role in many other functions such as DNA synthesis, replication, apoptosis, necrosis, and cancer progression. Herein, we review the current state of the PARP-1 role in DNA damage repair and other biological processes including epithelial to mesenchymal transition (EMT). We have also observed the role of PARP-1 in modulating EMT regulators like E-cadherin, Vimentin, Claudin-1, Snail, Smad-4, Twist-1, and ß-catenin. Here, we have also attempted to relate the role of PARP-1 in EMT of cancer cells.

Assessment of telomerase as drug target in breast cancer.

Telomerase is a specialized enzyme which maintains telomere length at the extreme end of the chromosome. Over 90% of all cases of cancer show high expression of telomerase while in normal cells, its expression is extremely low or undetectable. Detection of telomerase activity in a wide range of breast cancer makes telomerase an interesting target for diagnosis and therapy. In this review, we have aimed to describe telomerase as a therapeutic and accurate diagnostic target in breast cancer. Telomerase performs many extracurricular activities apart from maintaining telomere length; here, we have also tried to address its role in epithelialmesenchymal transition (EMT) of breast cancer progression.

CdSe- Reduced graphene oxide nanocomposite toxicity alleviation via V2O5 shell formation over CdSe core: in vivo and in vitro studies.

The present article demonstrates the synthesis of the nanocomposite of reduced graphene oxide (rGO) with CdSe and CdSe/V2O5 core/shell quantum dots by a two-step facile synthesis approach and subsequently studies their relative biocompatibility in different cells. Various characterization techniques have been applied including transmission electron microscopy (TEM), an x-ray diffractometer (XRD) and Raman spectroscopy to confirm the successful formation of CdSe-rGO and CdSe/V2O5-rGO nanocomposites. The average sizes of CdSe and CdSe/V2O5 QDs have found to be ∼3 and 5.5 nm, respectively with a good dispersion over the surface of rGO nanosheets. A crystal phase change has occurred during the formation of the V2O5 shell over the surface of CdSe QDs and confirmed through XRD. Raman spectroscopy has shown some useful insight of the surface state of CdSe and consequent changes in the surface with V2O5 shell growth. Further, MTT and cell growth assays have been performed to analyze their biocompatibility in A549 and Hela cells with various concentrations of as-synthesized materials. Our results demonstrate the toxicity of CdSe-rGO nanocomposite to be substantially reduced by the growth of the V2O5 shell. The in vivo studies in Drosophila show a remarkable decrease in the reactive oxygen species (ROS) and apoptosis levels for a CdSe/V2O5-rGO composite as compared to a CdSe-rGO nanocomposite, which paves a promising pathway for the CdSe/V2O5-rGO nanocomposite to be used as an efficient biocompatible material.

Measles virus phosphoprotein inhibits apoptosis and enhances clonogenic and migratory properties in HeLa cells.

Measles virus is the causative agent of measles, a major cause of child mortality in developing countries. Two major proteins, coded by the viral genome, are nucleocapsid protein (N) and phosphoprotein (P). The N protein protects the viral genomic RNA and forms ribonucleoprotein complex (RNP) together with P protein. MeV-P protein recruits the large protein (L), i.e. viral RNA-depended RNA polymerase (RdRp), to ensure viral replication in host cell. Apoptogenic properties of N protein of Edmonston vaccine strain have been established in our lab previously. We investigated the role of MeV-P protein of Edmonston vaccine strain as modulator of apoptosis in cervical cancer cell line (HeLa) and found that MeV-P protein is anti-apoptotic and enhances cell proliferation. Measles virus is considered to be innately oncotropic virus. However, the anti-apoptotic property of MeV-P protein raises important concerns while adopting this virus as an anti-cancer therapeutic tool.

Diversity and functional evolution of the plasminogen activator system.

The urokinase plasminogen activator system is a family of serine proteases which consists of uPA (urokinase plasminogen activator), uPAR (urokinase type plasminogen activator receptor) and PAI-1 (plasminogen activator inhibitor 1). In addition to their significant roles in activation, these proteases act as key regulators of the tumor microenvironment and are involved in the metastatic process in many cancers. High levels of uPA system proteases in many human cancer predicts poor patient prognosis and strongly indicated a key role of uPA system in cancer metastasis. Individual components of uPA system are found to be differentially expressed in cancer cells compared to normal cells and therefore are potential therapeutic targets. In this review, we present the molecular and cellular mechanisms underlying the role of uPA system in cancer progression. Epithelial to mesenchymal transitions (EMT) is the main cause of the cancer cell metastasis. We have also attempted to relate the role of uPA signaling in EMT of cancer cells.

Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells.

Reverse transcriptase activity of telomerase adds telomeric repeat sequences at extreme ends of the newly replicated chromosome in actively dividing cells. Telomerase expression is not detected in terminally differentiated cells but is noticeable in 90% of the cancer cells. hTERT (human telomerase reverse transcriptase) expression seems to promote invasiveness of cancer cells. We here present proteomic profiles of cells overexpressing or knocked down for hTERT. This study also attempts to find out the potential interacting partners of hTERT in cancer cell lines. Two-dimensional gel electrophoresis (2-DE) of two different cell lines U2OS (a naturally hTERT negative cell line) and HeLa revealed differential expression of proteins in hTERT over-expressing cells. In U2OS cell line 28 spots were picked among which 23 spots represented upregulated and 5 represented down regulated proteins. In HeLa cells 21 were upregulated and 2 were down regulated out of 23 selected spots under otherwise identical experimental conditions. Some heat shock proteins viz. Hsp60 and Hsp70 and GAPDH, which is a housekeeping gene, were found similarly upregulated in both the cell lines. The upregulation of these proteins were further confirmed at RNA and protein level by real-time PCR and western blotting respectively.

Global expression profile of telomerase-associated genes in HeLa cells.

Telomerase is a specialized nucleoprotein enzyme complex that maintains the telomere length. The telomerase reverse transcriptase (TERT) is the catalytically active component of the telomerase complex. In humans, the protein component (hTERT) and RNA component (hTR) are found to differentially express in cancer cells. In contrast to differentiated cells, most of the cancer cells overexpress hTERT, which is needed to maintain the proliferative potential of cells. The overexpression of telomerase is not proportionate to telomere length in cancer cells, suggesting that the immortalizing phenotype can be mediated through other factors in addition to telomere length. To investigate the role of hTERT in immortalizing process, loss of gene function studies were carried out. Short interfering RNA (siRNA) and short hairpin RNA (shRNA) against hTERT showed the reduction of hTERT transcript, reduction of telomerase activity and alteration of gene expression in HeLa cells. The molecular basis of proliferative capacity of hTERT was investigated by gene expression microarray. Analysis of microarray data for HeLa cells following siRNA and shRNA mediated knockdown of hTERT showed that 80 genes were upregulated and 73 genes downregulated. Out of these, 37 genes are known to be involved in cancer. Further analyses of previously known genes involved in cancer like KLF4, FGF2, IRF-9 and PLAU by Real Time PCR showed their upregulation. We are documenting for the first time the effect of knocking down hTERT on expression of KLF4 and FGF2. Interestingly, it has been earlier reported that KLF4 and FGF2 up-regulate the expression of hTERT in cancer cells. This suggests that hTERT may be subject to its own auto-regulatory effects.

Telomerase and its extracurricular activities.

The classical activity of telomerase is to synthesize telomeric repeats and thus maintain telomere length, which in turn ensures chromosome stability and cellular proliferation. However, there is growing evidence that implicates telomerase in many other functions that are independent of TERC being used as its template. Telomerase has an RNA-dependent RNA polymerase (RdRP) activity in the mitochondria. Other than viral RdRPs, it is the only RNA-dependent RNA polymerase that has been identified in mammals. It also plays a role in the Wnt signaling pathway by acting as a transcriptional modulator. Telomerase acts as a reverse transcriptase independent of its core subunit, TERC. Studies indicate that telomerase is also involved in apoptosis and DNA repair.