The RSC complex localizes to coding sequences to regulate Pol II and histone occupancy.

ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions.

Species Composition and Diversity Dynamics of Actinomycetes in Arid and Semi-arid Salt Basins of Rajasthan.

Species composition and diversity dynamics of the actinomycetes was studied in five salt basins of arid and semi-arid areas of Rajasthan, India. A novel approach integrating molecular (16S rRNA gene) and diversity indices was applied to reveal species composition and diversity dynamics. Fifty-three actinomycetes isolates were isolated from five arid and semi-arid salt basins. Molecular characterization resulted in the identification of actinomycetes species belonging to three genera namely, Streptomyces, Nocardiopsis, and Actinoalloteichus. The diversity study among actinomycetes species validates their universal occurrence in arid and semi-arid regions of Rajasthan. The species N. dassonvillei subsp. albirubida was omnipresent in all the five salt basins but its relative manifestation was not static across habitats. The study revealed that three species N. chromatogenes, S. durbertensis, and S. mangrovicola are being reported for the first time from India. The maximum species of actinomycetes were recorded from Pachpadra (14) and the minimum from Didwana area (6). This study not only documents the hitherto wealth of actinomycetes species in arid and semi-arid salt basins of Rajasthan but also reveals the composition and diversity dynamics of actinomycetes.

Detouring of cisplatin to access mitochondrial genome for overcoming resistance.

Chemoresistance of cisplatin therapy is related to extensive repair of cisplatin-modified DNA in the nucleus by the nucleotide excision repair (NER). Delivering cisplatin to the mitochondria to attack mitochondrial genome lacking NER machinery can lead to a rationally designed therapy for metastatic, chemoresistant cancers and might overcome the problems associated with conventional cisplatin treatment. An engineered hydrophobic mitochondria-targeted cisplatin prodrug, Platin-M, was constructed using a strain-promoted alkyne-azide cycloaddition chemistry. Efficient delivery of Platin-M using a biocompatible polymeric nanoparticle (NP) based on biodegradable poly(lactic-co-glycolic acid)-block-polyethyleneglycol functionalized with a terminal triphenylphosphonium cation, which has remarkable activity to target mitochondria of cells, resulted in controlled release of cisplatin from Platin-M locally inside the mitochondrial matrix to attack mtDNA and exhibited otherwise-resistant advanced cancer sensitive to cisplatin-based chemotherapy. Identification of an optimized targeted-NP formulation with brain-penetrating properties allowed for delivery of Platin-M inside the mitochondria of neuroblastoma cells resulting in ∼17 times more activity than cisplatin. The remarkable activity of Platin-M and its targeted-NP in cisplatin-resistant cells was correlated with the hyperpolarization of mitochondria in these cells and mitochondrial bioenergetics studies in the resistance cells further supported this hypothesis. This unique dual-targeting approach to controlled mitochondrial delivery of cisplatin in the form of a prodrug to attack the mitochondrial genome lacking NER machinery and in vivo distribution of the delivery vehicle in the brain suggested previously undescribed routes for cisplatin-based therapy.

Mce4A protein of Mycobacterium tuberculosis induces pro inflammatory cytokine response leading to macrophage apoptosis in a TNF-α dependent manner.

Mycobacterium tuberculosis subverts the host immune response through numerous immune-evasion strategies. Apoptosis has been identified as one such mechanism and has been well studied in M. tuberculosis infection. Here, we demonstrate that the Mce4A protein of mce4 operon is involved in the induction of host cell apoptosis. Earlier we have shown that the Mce4A was required for the invasion and survival of M. tuberculosis. In this report we present evidence to establish a role for Mce4A in the modulation of THP-1 cell survival. Recombinant Mce4A was expressed and purified from Escherichia coli as inclusion bodies and then refolded. Viability of THP-1 cells decreased in a dose-dependent manner when treated with Mce4A. The secretion of pro-inflammatory cytokines like tumor necrosis factor (TNF-α) or interferon gamma (IFN-γ), and enhanced nitric oxide release was observed when the THP-1 cells, were treated with Mce4A protein. The Mce4A induced apoptosis of the THP-1 cells was TNF-α dependent since blocking with anti TNF-α antibody abrogated this phenomenon. Collectively, these data suggest that Mce4A can induce the THP-1 cells to undergo apoptosis which primarily follows a TNF- α dependent pathway.

Unique Use of Alkylation for Chemo-Redox Activity by a Pt(IV) Prodrug.

Resistance towards chemotherapeutics displayed by cancer cells is a significant stumbling block against fruitful cisplatin-based therapy. A unique dual-acting chemotherapeutic modality, Platin-B, a prodrug of cisplatin and pipobroman-mimicking alkylating agent, was constructed to circumvent tumor resistance. Platin-B exhibited a superior cytotoxicity profile in cisplatin-resistant cancer cells. Enhanced activity and the ability to overcome cancer-induced resistance of Platin-B was related to adduct formation with intracellular glutathione, followed by the activity of Platin-B on the mitochondria of cells, along with its conventional nuclear activity. Alkylating moieties present on Platin-B enhanced its cellular and subcellular concentration and protected it from early drug sequestration by biological thiols.

Occurrence and characterization of hitherto unknown Streptomyces species in semi-arid soils.

Streptomyces the predominant genus of Actinobacteria and plays an important role in the recycling of soil organic matter and production of important secondary metabolites. The occurrence and diversity assessment of Streptomyces species revealed alkaline and poor nutrient status of soils of semi-arid region of Jodhpur, Rajasthan. The morphological and biochemical characterization of 21 Streptomyces isolates facilitated Genus level identification but were insufficient to designate species. Species designation based on 16S rRNA gene delineated 21 isolates into 14 Streptomyces species. Upon BLAST search, the test isolates exhibited 98 to 100% identities with that of the best aligned sequences of the NCBI database. The GC content of 16S rRNA gene sequences of all the Streptomyces isolates tested ranged from 59.03% to 60.94%. The multiple sequence alignment of all the 21 Streptomyces isolates generated a phylogram with high bootstrap values indicating reliable grouping of isolates based on nucleotide sequence variations by way of insertion, deletion and substitutions and 16S rRNA length polymorphism. Some of the Streptomyces species molecularly identified under present study are reported for the first time from semi-arid region of Jodhpur.

A Nanoparticle Cocktail: Temporal Release of Predefined Drug Combinations.

A single magic bullet is not enough for treatment of metastatic cancers. However, administration of a combination of free drugs can be extremely challenging because of the inability to control the correct choice of dosages and definitive delivery of the effective drug ratio at the target tissue due to the differences in pharmacokinetics and biodistribution of individual drugs. Here we report an engineered biodegradable polymer containing combination therapeutics that can be self-assembled into a controlled release nanoparticle with abilities to deliver multiple therapeutics in a predefined ratio following temporal release patterns. This platform technology can lead to a rationally designed combination therapy.

Association of growth related seedling traits in Acacia senegal under arid environment of western Rajasthan.

Climatic models and predictions indicate increase in aridity world over due to global warming. Arid environments occupy about one third land area of the world. A. senegal is the most important dryland resource of western Rajasthan desert ecosystem. The seeds of 13 low and high seed yielding exotic and indigenous provenances were evaluated for diversity and interrelationship among growth related seedling traits targeting establishment and end use of this species. Under the present study most of the growth related seedling traits varied within and amongst provenances. Highly significant correlation of dry biomass per plant of more than 72% with root length (73.3%), collar diameter (72.2%), shoot dryweight (99.7%), root dry weight (95.7%) and seedling length (79.9%) under the present study may be used for early selection. Similarly, highly significant positive correlation of seedling length with seven out of 12 growth related seedling traits validate strong inherent association of these traits under strong genetic control and are amenable for selection. Significant negative correlation in number of nodules per plant with root/shoot length ratio (-57.6%) and no correlation with 10 out of 12 growth related seedling traits tested advocate emphasis on other growth related seedling traits in selection of elite A. senegal genotypes for afforestation. The non significant associations suggest that per cent germination was independent of other characters and could be selected separately.

Copper-free click-chemistry platform to functionalize cisplatin prodrugs.

The ability to rationally design and construct a platform technology to develop new platinum(IV) [Pt(IV)] prodrugs with functionalities for installation of targeting moieties, delivery systems, fluorescent reporters from a single precursor with the ability to release biologically active cisplatin by using well-defined chemistry is critical for discovering new platinum-based therapeutics. With limited numbers of possibilities considering the sensitivity of Pt(IV) centers, we used a strain-promoted azide-alkyne cycloaddition approach to provide a platform, in which new functionalities can easily be installed on cisplatin prodrugs from a single Pt(IV) precursor. The ability of this platform to be incorporated in nanodelivery vehicle and conjugation to fluorescent reporters were also investigated.

Effect of co-inoculations of native PGPR with nitrogen fixing bacteria on seedling traits in Prosopis cineraria.

Prosopis cineraria significantly contribute to sand dune stabilization, soil fertility rejuvenation and is an integral component of agro-forestry systems in arid regions of India. Effect of different rhizobacterial seed treatments on seed germination and seedling traits in two genotypes of P. cineraria (HPY-1) and (FG-1) were tested. Observations on seed germination (%) and seedling traits viz., root length (cm), shoot length (cm), seedling weight (g) and seedling length of different treatments were recorded. Whereas, germination index (GI), seedling vigour index (SVI) and root/shoot length ratio were derived from the observed data. The scarification treatment with sulphuric acid for 10 minutes substantially enhanced germination from < 20% to 80-82% in control treatments. Treatments with co-inoculations of Bacillus licheniformis and Sinorhizobium kostiense or S. saheli supported the maximum seed germination and seedling growth and vigour. The maximum germination per cent (92.5%), seedling length (10.94 cm), seedling vigour index (10.12) and germination index (7.97) were recorded with treatment (V2T6) wherein seeds of high pod yielding genotype were co-inoculated with Bacillus licheniformis and S. kostiense. The higher positive correlations of seedling length v/s shoot length followed by SVI v/s seedling length, SVI v/s root length and seedling length v/s root length is a fair indicative of inter dependency of these characteristics. Higher R2 values of root length v/s shoot length followed by that of SVI v/s GI indicates that a regression line fits the data well and future outcomes of observed seedling traits are likely to be predicted by the model.

The prodrug platin-A: simultaneous release of cisplatin and aspirin.

Cancer-associated inflammation induces tumor progression to the metastatic stage, thus indicating that a chemo-anti-inflammatory strategy is of interest for the management of aggressive cancers. The platinum(IV) prodrug Platin-A was designed to release cisplatin and aspirin to ameliorate the nephrotoxicity and ototoxicity caused by cisplatin. Platin-A exhibited anticancer and anti-inflammatory properties which are better than a combination of cisplatin and aspirin. These findings highlight the advantages of combining anti-inflammatory treatment with chemotherapy when both the drugs are delivered in the form of a single prodrug.

Ethno-geographic distribution and histopathological classification of nasopharyngeal carcinoma in a single center in Nepal.

INTRODUCTION: Nasopharyngeal carcinoma (NPC) shows geographic and ethnic variation with specific etiopathogenesis. This study characterized the distribution of NPC patients stratified by ethnicity, geography, and histology in a tertiary-level cancer center in Nepal. METHODS: A descriptive retrospective study was designed to analyze NPC cases from different regions among patients visiting the hospital from 2016 to 2021. Demographic and clinical information was obtained from medical records. Ethical approval was granted by the Nepal Health Research Council (NHRC). Data analyses and visualization were carried out with R software. RESULTS: During the six-year period, a total of 65 individuals were diagnosed with NPC, comprising 42 males and 23 females. Patient median age was 43 years (range 11-85 years). A bimodal age distribution of cases was observed with peaks in patients aged 30-39 years and 50-59 years. Of the NPC patients studied, 29 were from Koshi Province, with 7 cases from Ilam district and 6 cases from Morang district. There were 18 patients in Bagmati Province, and Kathmandu district had the highest number of cases within this region, with 8 patients. The highest proportion of cases were observed among patients of Janajati ethnicity (60%), including Rai, Limbu, and Sherpa people. Histologically, undifferentiated non-keratinizing NPC was the most commonly observed subtype, accounting for 43.1% of cases, followed by 20% differentiated non-keratinizing NPC and 4.6% keratinizing NPC across the entire sample population. The majority of patients (75.3%) were diagnosed at an advanced stage (stage III or IV) with none diagnosed at stage I. CONCLUSIONS: In our study, most cases of NPC occurred in patients from provinces in eastern Nepal (Koshi province), and of the Janajati ethnic community. The most common histological subtype was undifferentiated non-keratinizing carcinoma. Further epidemiological studies could address differences in prevalence and the challenge of late presentation of NPC patients in Nepal.

Quinoline driven fluorescence turn on 1,3-bis-calix[4]arene conjugate-based receptor to discriminate Fe3+ from Fe2+.

The synthesis and characterization of a triazole linked quinoline appended calix[4]arene conjugate, L, and its fluorescence turn on receptor property for Fe(3+) have been demonstrated. The selective and sensitive discrimination of Fe(3+) has been shown using fluorescence and absorption titration experiments. The Fe(3+) binding to L has been further shown by ITC and ESI MS. The mode of binding of Fe(3+) by calix[4]arene conjugate has been shown by absorption, (1)H NMR and visual color change and the species were modeled based on DFT computations. The {L + Fe(3+)} has been shown to label cells with fluorescence imaging. Moreover the utility of this conjugate has been demonstrated by the combination logic gate system.

Development of a novel PCR restriction analysis of the hsp65 gene as a rapid method to screen for the Mycobacterium tuberculosis complex and nontuberculous mycobacteria in high-burden countries.

The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.

Synthetically Tunable Suprahybrid Nanoparticle Platform for the Efficacious Delivery of Therapeutics.

The discovery of lipid-hybrid nanosystems has offered potential solutions to various drug delivery and theranostic challenges. However, in many instances, the commonly used lipids and other components in these systems often pose challenges related to their solubility, physicochemical properties, immune compatibility, and limited synthetic tunability. In this work, we introduce a synthetically tunable supramolecular scaffold with amphiphilic characteristics based on the calix[4]arene macrocyclic system. We designed and synthesized two novel calix[4]arene-polyethylene glycol (PEG) conjugates, termed Cal-P1 and Cal-P2, and these were characterized utilizing a wide range of spectroscopic and analytical methods. The rational design of Cal-P1 and Cal-P2 demonstrates their utility in forming stable blended nanospheres with sustained drug release characteristics. The synergistic blending of PLGA and the calixarene scaffold (Cal-P1 and Cal-P2) in constructing long-lasting and controlled-release nanoparticles (NPs), which are optimized for encapsulating Nile Red dye, and their successful internalization and retention in HeLa cancer cells are demonstrated through in vitro assays. The potential of these NPs as sustained therapeutic carriers is investigated in vivo, showing improved retention compared to free dye with negligible toxicity. The successful design and construction of Cal-P1 and Cal-P2 nanosystems represent a new paradigm for addressing drug loading challenges, opening up opportunities for the development of highly efficient, synthetically tunable alternative adjuvants for drug encapsulation and delivery.

Multiple sensor array of Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ complexes of a triazole linked imino-phenol based calix[4]arene conjugate for the selective recognition of Asp, Glu, Cys, and His.

The triazole linked o-imino phenol appended calix[4]arene conjugate (L) has been synthesized and characterized. The structure of L has been established based on single crystal XRD. The binding and recognition behavior of conjugate, L toward the transition metal ions, such as Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+), has been demonstrated using fluorescence, absorption and ESI-MS techniques. The in situ prepared complexes of these metal ions, namely, [Mn(2)L], [Fe(2)L], [Co(2)L], [Ni(2)L], [Cu(2)L], and [Zn(2)L] have shown recognition toward Glu, Asp, His and Cys. Hence L provides a multiple sensing molecular tool where the response for the recognition of biologically active amino acids of metalloproteins is elicited by the presence of specific metal ion.

Pyrophosphate sensing by a fluorescent Zn2+ bound triazole linked imino-thiophenyl conjugate of calix[4]arene in HEPES buffer medium: spectroscopy, microscopy, and cellular studies.

An in situ prepared Zn(2+) complex of triazole linked imino-thiophenyl conjugate of calix[4]arene, [ZnL], was demonstrated to be highly fluorescent in HEPES buffer solution. [ZnL] has been used as a chemo-sensing ensemble for the recognition of phosphates in general and pyrophosphates in particular among the eighteen different anions studied. The chemo-sensing behavior of the [ZnL] has been demonstrated through fluorescence, absorption, visual fluorescent color changes, ESI MS, and (1)H NMR titrations. Variations in the microstructural features of L, its zinc complex and the complex upon addition of PPi have been demonstrated through atomic force microscopy and transmission electron microscopy. Such studies have been extended to see the permeability of the conjugate into the HeLa cells by fluorescence microscopy. In accession, a reversible "write-read-erase-read" logic gate property of L has been demonstrated through a feedback loop in the presence of Zn(2+) and PPi.

Cd2+ complex of a triazole-based calix[4]arene conjugate as a selective fluorescent chemosensor for Cys.

An N,N-Dimethylamine ethylimino-appended triazole-linked calix[4]arene conjugate, L, has been synthesized and characterized, and its Cd(2+) complex has been isolated and characterized. The structure of [CdL] was established by computational calculation using B3LYP/LANL2DZ. Time-dependent density functional theory calculations were performed to demonstrate the electronic properties of [CdL]. This highly fluorescing [CdL] has been used to recognize Cys selectively among the 20 naturally occurring amino acids. [CdL] exhibits a minimum detection limit of 58 ppb for Cys, with reusability and reversibility being imparted to the system during sensing. Thus, the sensing of Cys was well demonstrated using various techniques, viz., fluorescence, absorption, visual color change, electrospray ionization MS, (1)H NMR, and live cell imaging experiments.

A Zn2+ specific triazole based calix[4]arene conjugate (L) as a fluorescence sensor for histidine and cysteine in HEPES buffer milieu.

A highly fluorescent Zn(2+) complex of the triazole linked salicyl-imino-thiophenyl conjugate of calix[4]arene, [ZnL] has been demonstrated to be a chemo-sensing ensemble for the recognition of His and Cys among the naturally occurring amino acids in HEPES buffer milieu. The recognition behaviour of the [ZnL] towards these amino acids has been shown on the basis of fluorescence, absorption and visual fluorescent colour changes. The species of recognition were shown by ESI MS titrations, AFM & TEM microscopy and cell studies.

Imino-phenolic-pyridyl conjugates of calix[4]arene (L1 and L2) as primary fluorescence switch-on sensors for Zn2+ in solution and in HeLa cells and the recognition of pyrophosphate and ATP by [ZnL2].

Pyridyl-based triazole-linked calix[4]arene conjugates, viz. L(1) and L(2), were synthesized and characterized. These two conjugates were shown to be selective and sensitive for Zn(2+) among the 12 metal ions studied in HEPES buffer medium by fluorescence, absorption, and visual color change with the detection limit of ~31 and ~112 ppb, respectively, by L(1) and L(2). Moreover, the utility of the conjugates L(1) and L(2) in showing the zinc recognition in live cells has also been demonstrated using HeLa cells as monitored by fluorescence imaging. The zinc complexes of L(1) and L(2) were isolated, and the structure of [ZnL(1)] has been established by single-crystal XRD and that of [ZnL(2)] by DFT calculations. TDDFT calculations were performed in order to demonstrate the electronic properties of receptors and their zinc complexes. The isolated zinc complexes, viz. [ZnL(1)] and [ZnL(2)], have been used as molecular tools for the recognition of anions on the basis of their binding affinities toward Zn(2+). [ZnL(2)] was found to be sensitive and selective toward phosphate-bearing ions and molecules and in particular to pyrophosphate (PPi) and ATP among the other 18 anions studied; however, [ZnL(1)] was not sensitive toward any of the anions studied. The selectivity has been shown on the basis of the changes observed in the emission and absorption spectral studies through the removal of Zn(2+) from [ZnL(2)] by PPi. Thus, [ZnL(2)] has been shown to detect PPi up to 278 ± 10 ppb at pH 7.4 in aqueous methanolic (1/2 v/v) HEPES buffer.