A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

Influence of climatic factors on the life stages of Aedes mosquitoes and vectorial transmission: A review.

BACKGROUND OBJECTIVES: Aedes aegypti and Aedes albopictus are two sympatric mosquito species that compete with each other for resources when their breeding habitats overlap. This study examines what happens when sympatric Aedes aegypti and Aedes albopictus mosquitoes' mate with each other and other species by looking at insemination rates, fecundity, and hatchability rate. METHODS: We performed controlled mating experiments in laboratory setting, assessing both conspecific and interspecific crosses. We measured insemination rates, egg numbers, and hatching success to examine the reproductive interference dynamics between these two distinct mosquito species. RESULTS: In the context of conspecific mating, it was observed that both female Ae. aegypti and Ae. albopictus exhibited high insemination rates, with percentages of 98% and 94%, respectively. However, interspecific mating exhibited interesting asymmetries: Ae. albopictus males achieved a notable insemination success rate of 28% when mating with Ae. aegypti females, while Ae. aegypti males achieved only 8% insemination success with Ae. albopictus females. Additionally, females that mated with interspecific males had reduced production of viable eggs compared to conspecific mating. Most notably, interspecific mating resulted in the production of infertile eggs, while conspecific mating led to successful hatching. INTERPRETATION CONCLUSION: The study reveals that, Ae. aegypti and Ae. albopictus can asymmetrically interfere with each other's reproduction, causing a 'satyr' effect. This understanding of interspecific competition and reproductive interference in these mosquito species could impact their coexistence in shared breeding habitats.

Susceptibility status of Aedes aegypti (Diptera: Culicidae) against insecticides of public health use in Delhi and NCR region, India.

BACKGROUND & OBJECTIVES: Aedes (Stegomyia) aegypti is a primary vector responsible for the transmission of various arboviral diseases in India. Without an effective drug or vaccine against these diseases, chemical insecticide-based vector control supplemented with source reduction remains the best option for their effective management. The development of insecticide resistance due to the continuous use of insecticides might affect the control operations. METHODS: Adults and larvae of Aedes aegypti were collected from different localities in Delhi. Larvae were exposed to discriminating (0.02mg/l) and application (1mg/l) doses of temephos. WHO tube assay was conducted for F1 adults using impregnated insecticide papers of dichlorodiphenyltrichloroethane (DDT), malathion, deltamethrin, permethrin, cyfluthrin, and lambda-cyhalothrin. RESULTS: Larvae of Ae. aegypti were found resistant (76.0%) to the discriminating dose of temephos, whereas suscep-tible (100.0%) to the application dose of the temephos. Adult Aedes (Fl) mosquitoes were resistant to DDT (23.7%), malathion (90.5%), deltamethrin (76.0%), permethrin (96.2 %) cyfluthrin (85.5%), and lambda-cyhalothrin (94.0%). INTERPRETATION & CONCLUSION: Indoor residual spray is not used in Delhi for vector control. Resistance in Aedes might be due to pesticide usage for agricultural activities in peripheral regions of Delhi. There is a need to investigate more on the insecticide resistance mechanisms for indirect resistance development. Understanding the insecticide susceptibility status of urban vectors is critical for planning effective control strategies.

Aedes aegypti container preference for oviposition and its possible implications for dengue vector surveillance in Delhi, India.

OBJECTIVES: Dengue is a mosquito-borne viral disease globally transmitted by Aedes aegypti. The most effective method to prevent the transmission of the disease is proficient vector control. Understanding the breeding behaviour of the responsible vectors is very pertinent in this regard; therefore, the present study was conducted to understand Ae. aegypti behaviour regarding the selection of containers for oviposition in the megacity of Delhi. METHODS: A household survey in different localities within Delhi was carried out during 2018-2019. All available containers were inspected for the presence of immature Ae. aegypti. In entomological surveillance, the ovipositional preference of Aedes was computed using the breeding preference ratio, container index in the field, and laboratory settings, and associations of dengue cases with monthly variation in environmental factors and container type were also calculated. RESULTS: The household larval survey in 40 localities showed that 40% of 27,776 water-holding containers in 3,400 houses were plastic, followed by overhead tanks (26.2%), and coolers (12.1%). The most preferred breeding habitat was clay pots (9.3%), followed by metallic containers (8.5%) and solid waste (7.1%). A laboratory-based study showed that Aedes preferred clay containers (81.8%) over 4 other types of containers (plastic, paper, metal, and glass). CONCLUSIONS: The present study provides a rationale for using clay containers as a possible surveillance tool (ovitraps) or as a vector control tool. This information might aid researchers in developing novel traps and targeting preferred containers for larval control activities during transmission and non-transmission seasons.

AST-487 Inhibits RET Kinase Driven TERT Expression in Bladder Cancer.

Mutations in the promoter of the human Telomerase Reverse Transcriptase (hTERT) gene are common and associated with its elevated expression in bladder cancer, melanoma, and glioblastoma. Though these mutations and TERT overexpression are associated with aggressive disease and poor outcome, an incomplete understanding of mutant TERT regulation limits treatment options directed at this gene. Herein, we unravel a signaling pathway that leads to upregulated hTERT expression resulting from the -124 bp promoter mutation, the most frequent variant across human cancer. We employed engineered bladder cancer cells that harbor a GFP insertion at the TSS region on -124 hTERT promoter for high-content screening drug discovery using a focused library of ~800 kinase inhibitors. Studies using in vitro and in vivo models prioritized AST-487, an inhibitor of the wild-type, and mutant RET (rearranged during transfection) proto-oncogene as a novel drug inhibitor of both wild-type and mutant promoter-driven hTERT expression. We also identified the RET kinase pathway, targeted by AST-487, as a novel regulator of mutant hTERT promoter-driven transcription in bladder cancer cells. Collectively, our work provides new potential precision medicine approaches for cancer patients with upregulated hTERT expression, perhaps, especially those harboring mutations in both the RET gene and the hTERT promoter, such as in thyroid cancer.

Live Cell Imaging of Nuclear Actin Filaments and Heterochromatic Repair foci in Drosophila and Mouse Cells.

Pericentromeric heterochromatin is mostly composed of repeated DNA sequences, which are prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that 'safe' homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins that drive directed motions. Understanding this pathway requires the detection of nuclear actin filaments that are significantly less abundant than those in the cytoplasm, and the imaging and tracking of repair sites for long time periods. Here, we describe an optimized protocol for live cell imaging of nuclear F-actin in Drosophila cells, and for repair focus tracking in mouse cells, including: imaging setup, image processing approaches, and analysis methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.

MiRNA182 regulates percentage of myeloid and erythroid cells in chronic myeloid leukemia.

The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.

Evaluation of hexane and ethyl acetate extracts of the sponge Jaspis diastra collected from Mauritius Waters on HeLa cells.

OBJECTIVES: Based on previous screening results, the cytotoxic effect of the hexane (JDH) and ethyl acetate extracts (JDE) of the marine sponge Jaspis diastra were evaluated on HeLa cells and the present study aimed at determining their possible mechanism of cell death. METHODS: Nuclear staining, membrane potential change, flow cytometry analysis of cell cycle distribution and annexin V staining were undertaken to investigate the effects of JDE and JDH. Electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance were used to characterize an isolated bioactive molecule. KEY FINDINGS: JDE displayed an IC50 25 times more significant than the JDH. Flow cytometry analysis revealed JDE induced apoptosis in HeLa cells accompanied by the collapse of mitochondrial membrane potential. Fractionation of JDE resulted in the isolation of the known cytotoxic cyclodepsipeptide, Jaspamide. CONCLUSIONS: Taking our results together suggest that JDE can be valuable for the development of anticancer drugs, especially for cervical cancer. Further investigations are currently in progress with the aim to determine and isolate other bioactive compounds from this extract.

Molecular epidemiology and virulence characteristics of prevalent group A streptococci recovered from patients in northern India.

INTRODUCTION: In this study, the prevalence of M types of Group A Streptococcus (GAS) in North India, invasive behavior of prevalent M types, and inflammatory immune response by host cells were studied. METHODOLOGY: A total of 1,047 clinical samples were collected between 2004 and 2010. Confirmation of GAS was determined by serotyping and M types were identified by emm gene sequencing. The most prevalent serotypes were selected to study their invasive behavior and inflammatory immune response under different temperatures and salt concentrations in A549 and HEp-2 cells. RESULTS: Ninety-two isolates were identified as GAS of which 17 were M types with 18.5% heterogeneity. The most prevalent M types were M1 (21.73%) and M49 (8.7%), respectively. M1 and M49 were used to study virulence potential and inflammatory immune responses. The efficiency of cell infection decreased with increased temperature for both M types, increasing with lowering temperatures compared to the uninfected control (37°C). As salt concentration was increased, cell infection efficiency was lowered with some exceptions; the infection efficiency of M1 strain in A549 cells with 0.6 M NaCl was 50 fold higher (p ≤ 0.03). Significantly increased production of IL-6 and IL-8 was observed in both cell lines infected with GAS and when grown under different environmental conditions compared to uninfected cell lines. CONCLUSIONS: This study determined the prevalence of different M types in North India and showed that environmental conditions can regulate cell infection by GAS . This information may influence the selection of GAS serotypes used in vaccine development.

Ethyl acetate extract of the Mauritian sponge Jaspis sp. induces cell arrest in human promyelocytic leukemia cells.

Marine sponges are considered as a gold mine of new natural products possessing numerous biological activities. We examined the cytotoxic properties of the ethyl acetate extract (JDE) of the previously unrecorded sponge, Jaspis sp. collected from Mauritius Waters. JDE displayed an interesting IC50 of 0.057±0.04µg/mL on HL-60 cells evaluated by MTS assay. Mitochondrial membrane potential change, microscopic analysis and DNA fragmentation assays also confirmed JDE induced apoptosis on HL-60 cells. Annexin V staining demonstrated that JDE induced apoptosis at different concentrations. Treatment with 100ng/mL of JDE led to an accumulation of cells in G2/M phase after 24 h, causing a significant increase of cells (24h: 5.84%; 48h: 13.41%) in sub-G1 phase suggesting that JDE can induce cell cycle arrest in G2/M phase.

Role of pilus proteins in adherence and invasion of Streptococcus agalactiae to the lung and cervical epithelial cells.

Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. We have performed comparative genomics of prevalent GBS serotypes of Indian origin (i.e. Ia, III, V, and VII). Pilus-proteins were commonly found up-regulated, and their expression was studied by using antiserum for GBS80 (backbone protein of pilus island-I), GBS67 (ancillary protein of PI-2a), and SAN1518 (backbone protein of PI-2b) by whole cell and Western blot analysis. To check the role of pilus proteins in adherence and invasion, an inhibition assay was performed. Comparative immunoblotting experiments revealed that expression of pili proteins does not differ in geographically different selected serotypes, Ia and V, of India and the United States. In the case of A549 cells, we found that GBS VII invasion and adherence was inhibited by pilus protein-specific antiserum SAN1518 significantly (p < 0.001) by 88.5 and 91%, respectively. We found that mutant strains, deficient in the pilus proteins (Δgbs80 and Δsan1518) exhibit a significant decrease in adherence in the case of type Ia, III, and VII. In the case of type VII, we have found a 95% reduction in invasion when Δsan1518 was used with A549 cells. Because the pilus proteins were identified previously as vaccine candidates against GBS serotypes of developed countries, we also found their role in the attachment and invasion of GBS of Indian origin. Thus, the present work supports the idea of making a more effective pilus protein-based vaccine that can be used universally.

Identification of potential universal vaccine candidates against group A Streptococcus by using high throughput in silico and proteomics approach.

Streptococcus pyogenes or group A Streptococcus (GAS) causes ~700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered due to high strain and serotype diversity in different geographical regions, and the generation of cross-reactive antibodies that may induce autoimmune disease. There is an urgent need to search for alternative vaccine candidates. High throughput multigenome data mining coupled with proteomics seems to be a promising approach to identify the universal vaccine candidates. In the present study, in silico analysis led to prediction of 147 proteins as universal vaccine candidates. Distribution pattern of these predicted candidates was explored in nonsequenced Indian GAS strains (n = 20) by using DNA array hybridization validating in silico analysis. High throughput analyses of surface proteins using 1D-SDS-PAGE coupled with ESI-LC-MS/MS was applied on highly (M49) and less (M1) invasive GAS strains of Indian origin. Comparative proteomics analysis revealed that highly invasive GAS M49 had metabolically more active membrane associated protein machinery than less invasive M1. Further, by overlapping proteomics data with in silico predicted vaccine candidate genes, 52 proteins were identified as probable universal vaccine candidates, which were expressed in these GAS serotypes. These proteins can further be investigated as universal vaccine candidates against GAS. Moreover, this robust approach may serve as a model that can be applied to identify the universal vaccine candidates in case of other pathogenic bacteria with high strain and genetic diversity.

Nano-technology for targeted drug delivery to combat antibiotic resistance.

Several microbes have evolved clinically significant resistance against almost every available antibiotic. Yet the development of new classes of antibiotics has lagged far behind our growing need. Frequent and suboptimal use of antibiotics particularly in developing countries aggravated the problem by increasing the rate of resistance. Therefore, developing new and multidimensional strategies to combat microbial infections is warranted. These include i) modification of existing antibiotics, ii) searching new and novel antibiotics, iii) development and improvement of antibiotics carrier system to reduce amount and frequency of antibiotic doses, iv) development of targeted antibiotic delivery systems. Here, the authors discuss trends and development of nano-materials and alternative antimicrobials to solve the problem of antibiotic resistance.