Recent Developments on Electroactive Organic Electrolytes for Non-Aqueous Redox Flow Batteries: Current Status, Challenges, and Prospects.

The ever-increasing threat of climate change and the depletion of fossil fuel resources necessitate the use of solar- and wind-based renewable energy sources. Large-scale energy storage technologies, such as redox flow batteries (RFBs), offer a continuous supply of energy. Depending on the nature of the electrolytes used, RFBs are broadly categorized into aqueous redox flow batteries (ARFBs) and non-aqueous redox flow batteries (NARFBs). ARFBs suffer from various problems, including low conductivity of electrolytes, inferior charge/discharge current densities, high-capacity fading, and lower energy densities. NARFBs offer a wider potential window and range of operating temperatures, faster electron transfer kinetics, and higher energy densities. In this review article, a critical analysis is provided on the design of organic electroactive molecules, their physiochemical/electrochemical properties, and various organic solvents used in NARFBs. Furthermore, various redox-active organic materials, such as metal-based coordination complexes, quinones, radicals, polymers, and miscellaneous electroactive species, explored for NARFBs during 2012-2023 are discussed. Finally, the current challenges and prospects of NARFBs are summarized.

Development of Membranes and Separators to Inhibit Cross-Shuttling of Sulfur in Polysulfide-Based Redox Flow Batteries: A Review.

The global rapid transition from fossil fuels to renewable energy resources necessitates the implementation of long-duration energy storage technologies owing to the intermittent nature of renewable energy sources. Therefore, the deployment of grid-scale energy storage systems is inevitable. Sulfur-based batteries can be exploited as excellent energy storage devices owing to their intrinsic safety, low cost of raw materials, low risk of environmental hazards, and highest theoretical capacities (gravimetric: 2600 Wh/kg and volumetric: 2800 Wh/L). However, sulfur-based batteries exhibit certain scientific limitations, such as polysulfide crossover, which causes rapid capacity decay and low Coulombic efficiency, thereby hindering their implementation at a commercial scale. In this review article, we focus on the latest research developments between 2012-2023 to improve the separators/membranes and overcome the shuttle effect associated with them. Various categories of ion exchange membranes (IEMs) used in redox batteries, particularly polysulfide redox flow batteries and lithium-sulfur batteries, are discussed in detail. Furthermore, advances in IEM constituents are summarized to gain insights into different fundamental strategies for attaining targeted characteristics, and a critical analysis is proposed to highlight their efficiency in mitigating sulfur cross-shuttling issues. Finally, future prospects and recommendations are suggested for future research toward the fabrication of more effective membranes with desired properties.

Recent Development of Electrolytes for Aqueous Organic Redox Flow Batteries (Aorfbs): Current Status, Challenges, and Prospects.

In recent years, aqueous organic redox flow batteries (AORFBs) have attracted considerable attention due to advancements in grid-level energy storage capacity research. These batteries offer remarkable benefits, including outstanding capacity retention, excellent cell performance, high energy density, and cost-effectiveness. The organic electrolytes in AORFBs exhibit adjustable redox potentials and tunable solubilities in water. Previously, various types of organic electrolytes, such as quinones, organometallic complexes, viologens, redox-active polymers, and organic salts, were extensively investigated for their electrochemical performance and stability. This study presents an overview of recently published novel organic electrolytes for AORFBs in acidic, alkaline, and neutral environments. Furthermore, it delves into the current status, challenges, and prospects of AORFBs, highlighting different strategies to overcome these challenges, with special emphasis placed on their design, composition, functionalities, and cost. A brief techno-economic analysis of various aqueous RFBs is also outlined, considering their potential scalability and integration with renewable energy systems.

Melamine-based hyper-cross-linked resin for efficient removal of total petroleum hydrocarbons (C10-C32) from aqueous solutions.

Petroleum hydrocarbons (PHCs) are produced from industrial discharges, storage leakages, accidental spills, and operational failures. The hazardous nature of PHCs causes serious health risks and threatens the entire aquatic habitat. In this research work, the investigation of the removal of total petroleum hydrocarbons (TPHs) from the contaminated water is carried out utilizing a novel hypercross-linked resin, MAICY, which is generated by condensation of commercially available precursors. The chemical structures of MAICY have been examined extensively by FESEM, FT-IR, solid (CP-MAS) 13C-NMR, and TGA. A comprehensive analysis for adsorption parameters of TPHs has been performed, and different models such as Langmuir and Freundlich isotherms have been employed where the Freundlich isotherm was found to be the best fit for removal of THPs (R2= 0.9991). The results revealed that the performance of MAICY for the adsorption of TPHs from contaminated water gives a maximum adsorption capacity (qe) of 146 mg.g-1. The results of various parameters hinted that the contact time (0.25-4 h), the dosage of adsorbent (0.17 g/L), pH (7), and concentration of TPHs (26.5 mg/L) have controlled the overall adsorptive performance. Moreover, the kinetic data of qe(expt.) and qe(calc.) for adsorption of TPHs disclosed the regression values (R2) for pseudo-first order (R2= 0.9921) and pseudo-second order (R2= 0.9891). Additionally, based on CHI factor (X2) error estimations, the data was shown to be more consistent with pseudo-first-order kinetics. Moreover, MAICY demonstrated excellent reusability and recycling properties for up to four consecutive adsorption-desorption cycles.

Synovial sarcoma: a rare neoplasm of paranasal sinus.

Synovial Sarcoma (SS) is a rare soft-tissue malignant tumour. Its presentation in the head and neck region is uncommon. Because of the complex anatomy of the head and neck region, surgery with clear margins is not achievable. In such cases, a multi-modality approach is required as there is no established standard of care. In this report, we share the case of a girl who presented with nasal obstruction. Imaging revealed a mass involving the left nasal cavity, paranasal sinuses without intracranial extension. It was diagnosed as synovial sarcoma. She underwent surgical excision and adjuvant radiation therapy (RT) to the tumour bed, followed by an incomplete course of chemotherapy. Later on, she developed systemic disease. Considering the rarity of this case and lack of guidelines for standard treatment, we report on this case to share our experience with management and treatment outcome.

Recent Trends and Future Perspectives of Emergent Analytical Techniques for Mercury Sensing in Aquatic Environments.

Environmental emissions of mercury from industrial waste and natural sources, even in trace amounts, are toxic to organisms and ecosystems. However, industrial-scale mercury detection is limited by the high cost, low sensitivity/specificity, and poor selectivity of the available analytical tools. This review summarizes the key sensors for mercury detection in aqueous environments: colorimetric-, electrochemical-, fluorescence-, and surface-enhanced Raman spectroscopy-based sensors reported between 2014-2021. It then compares the performances of these sensors in the determination of inorganic mercury (Hg2+ ) and methyl mercury (CH3 Hg+ ) species in aqueous samples. Mercury sensors for aquatic applications still face serious challenges in terms of difficult deployment in remote areas and low robustness, reliability, and selectivity in harsh environments. We provide future perspectives on the selective detection of organomercury species, which are especially toxic and reactive in aquatic environments. This review is intended as a valuable resource for scientists in the field of mercury sensing.

Optical Chemical Sensing of Iodide Ions: A Comprehensive Review for the Synthetic Strategies of Iodide Sensing Probes, Challenges, and Future Aspects.

Among several anions, iodide (I- ) ions play a crucial role in human biological activities. In it's molecular form (I2 ), iodine is utilized for several industrial applications such as syntheses of medicines, fabric dyes, food additives, solar cell electrolytes, catalysts, and agrochemicals. The excess or deficiency of I- ions in the human body and environmental samples have certain consequences. Therefore, the selective and sensitive detection of I- ions in the human body and environment is vital for monitoring their overall profile. Amongst various analytical techniques for the estimation of I- ions, optical-chemical sensing possesses the merits of high sensitivity, selectivity, and utilizing the least amount of sensing materials. The distinctive aims of this manuscript are (i) To comprehensively review the development of optical chemical sensors (fluorescent & colorimetric) reported between 2001-2021 using organic fluorescent molecules, supramolecular materials, conjugated polymers, and metal-organic frameworks (MOFs). (ii) To illustrate the design and synthetic strategies to create specific binding and high affinity of I- ions which could help minimize negative consequences associated with its large size and high polarizability. (iii) The challenges associated with sensitivity and selectivity of I- ions in aqueous and real samples. The probable future aspects concerning the optical chemical detection of I- ions have also been discussed in detail.

Photocatalytic Water-Splitting by Organic Conjugated Polymers: Opportunities and Challenges.

The future challenges associated with the shortage of fossil fuels and their current environmental impacts intrigued the researchers to look for alternative ways of generating green energy. Solar-driven water splitting into oxygen and hydrogen is one of those advanced strategies. Researchers have studied various semiconductor materials to achieve potential results. However, it encountered multiple challenges such as high cost, low photostability and efficiency, and required multistep modifications. The conjugated polymers (CPs) have emerged as promising alternatives for conventional inorganic semiconductors. The CPs offer low cost, sufficient light absorption efficiency, excellent photo and chemical stability, and molecular optoelectronic tunable characteristics. Furthermore, organic CPs also present higher flexibility to tune the basic framework of the backbone of the polymers, amendments in the sidechain to incorporate desired functionalities, and much-needed porosity to serve better for photocatalytic applications. This review article summarizes the recent advancements made in visible-light-driven water splitting covering the aspects of synthetic strategies and experimental parameters employed for water splitting reactions with special emphasis on conjugated polymers such as linear CPs, planarized CPs, graphitic carbon nitride (g-C3 N4 ), conjugated microporous polymers (CMPs), covalent organic frameworks (COFs), and conjugated polymer-based nanocomposites (CPNCs). The current challenges and future prospects have also been described briefly.

Isotopic Interpretation of Particulate Nitrate in the Metropolitan City of Karachi, Pakistan: Insight into the Oceanic Contribution to NOx.

Nitrogen oxide (NOx) abatement has become the focus of air quality management strategies. In this study, we examined NOx sources and the atmospheric conversion of NOx in Karachi, Pakistan, a megacity in South Asia with serious particulate pollution problems. Oceanic contributions to NOx were quantified for the first time based on a novel approach using nitrogen/oxygen isotopic analysis in nitrate (δ15N-NO3-; δ18O-NO3-) and a Bayesian model. Our results showed that δ15N-NO3- in Karachi varied between -10.2‰ and +12.4‰. As indicated by the δ18O-NO3- findings (+66.2 ± 7.8‰), the •OH pathway dominated NOx conversion throughout the nearly two-year observation, but high NO3- events were attributed to the O3 pathway. Coal combustion was the most significant source (32.0 ± 9.8%) of NOx in Karachi, with higher contributions in the autumn and winter; a similar situation occurred for biomass burning + lightning (30.3 ± 6.5%). However, mobile sources (25.2 ± 6.4%) and microbial processes (12.5 ± 7.5%) exhibited opposite seasonal trends. The oceanic contributions to NOx in Karachi were estimated to be 16.8%, of which lightning, shipping emissions, and microbial processes accounted for 20.3%, 46.3%, and 33.4%, respectively, emphasizing the dominance of shipping emissions as an oceanic NOx source.

Synthesis of a novel polysuccinimide based resin for the ultrahigh removal of anionic azo dyes from aqueous solution.

A novel hyper-cross-linked polyamide resin (PSI-PA) has been synthesized for the ultrahigh removal of Congo red (CR) and Eriochrom Black T (EBT) from aqueous solution. The mesoporous resin, having a specific surface area (98.80 m2 g-1), showed maximum adsorption capacity (Qmax) of 522.18 mg g-1 for CR (pH 9.0) and 460.34 mg g-1 for EBT (pH 6) at room temperature. The adsorption of these dyes was rapid and the equilibrium was attained within 4 h. The kinetic data was well-fitted by pseudo-second-order rate equation. Analysis of the surface chemical composition of loaded PSI-PA by XPS revealed the appearance of a new peak at around 166.0 eV (S 2p), confirming the adsorption of the sulfur-based dyes onto the resin. Examination of experimental data of dyes adsorption by a variety of non-linear adsorption isotherms and kinetic models suggested that the Langmuir model exhibited the best fit with high correlation coefficients for both CR (R2 = 0.9966) and EBT (R2 = 0.9934). PSI-PA has been extensively characterized by FT-IR, solid state 13C (CP-MAS) NMR, EDS, TGA and BET analysis. Moreover, PSI-PA exhibited 82% removal efficiency for dyes in simulated effluents, manifesting its promise and utility for treating industrial effluent.

First Report of Aspergillus niger causing Black rot of Grapes in Pakistan.

In June 2015 & 2016, a postharvest survey of table grapes (Vitis vinifera) cv. King's Ruby, was carried out in five different commercial fruit markets of Rawalpindi (33°38'19.2″N, 73°01'45.0″E) district, Punjab Province. Symptoms appeared as brownish lesions with black sporulation on grapes berries. The incidence of these symptoms on bunches ranged from 12 to 17% at all sites. Symptomatic tissue pieces were surface-sterilized with 0.1% sodium hypochlorite (NaOCl) for 30 seconds, rinsed three times with sterile distilled water, dried on filter paper for 45 seconds, and incubated on potato dextrose agar (PDA) at 25°C. After 3 days, dark brown to black mycelium were formed on PDA media. A total of 24 isolates were examined morphologically. The apex of the conidiophore was observed to be radiate. Vesicles were found to be spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose measured (3.14 µm ± 2.24 in averaged diameter: n=50), dark brown to black, with roughened cell walls. The conidiophores were also smooth-walled, hyaline, and became melanized toward the vesicle. These characteristics of the fungus were similar to those described for Aspergillus niger van Tiegh (de Hoog et al. 2000). For molecular identification, the internal transcribed spacer (ITS) region, beta-tubulin (Bt) gene and partial RNA polymerase II largest subunit (RPB2) gene of representative isolate (Asp.n02) was amplified using primers ITS1/ITS4, BT2a/BT2b and RPB2-6F/RPB2-7R respectively (White et al., 1990; Glass & Donaldson, 1995; Liu et al. 1999). Sequences were deposited in GenBank (ITS, MN658871; Bt2, MT117924; and RPB2, MT318289). Based on BLAST analysis, sequences of the ITS region, Bt2 genes, and RPB2 gene showed 99 to 100% similarity of isolate Asp.n02 to Aspergillus niger (Accession Nos. MK307680.1, MN195121.1, MF078661.1 for ITS gene, MN567299.1, MK451029.1, MK451020.1 for Bt2 gene, and MK450788.1, MK450790.1 for RPB2 gene). To complete Koch's postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of isolate: Asp.n 02 was pipetted onto three non-wounded and four wounded (5 mm diam) asymptomatic grape berries cv. King's Ruby (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries similaries to serve as a negative control (Ghuffar et al. 2018; Jayawardena et al. 2018). Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice. Brownish lesions leading to black sporulation similar to the original symptoms were observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control. The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Aspergillus niger was reported previously in Europe and Israel causing mycotoxin (Ochratoxin A) OTA production on Table grapes (Bau et al. 2006). To our knowledge, this is the first report of Aspergillus niger causing black rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against the black rot of grapes in Pakistan.

Potential risk factors of ovarian cancer and analysis of CA125, a biomarker used for its monitoring and diagnosis.

Ovarian cancer (OC) is the fourth most common cancer among Pakistani, Scottish and Chinese women. The aim of the present study was to determine the association of potential risk factors with OC and analysis of Cancer Antigen 125 (CA125) in its monitoring and diagnosis. A total of 200 patients diagnosed with OC were included in this study. All the patients were interviewed and 54 OC patients (case group) and 35 age-matched healthy subjects (control group) gave their blood for analysis of CA125. The blood of case and control groups was subjected to an ELISA test for the evaluation of CA125 levels. Majority of the patients were of 40-50 years of age and most of the patients were diagnosed at this period of life. The majority of the patients experienced their first menarche and menopause at the age of 13-14 and 40-50 years respectively. There is no significant association between early menarche and OC family history, nor between late menopause and OC family history. There is a significant association between family history of breast cancer (BC) and age of menarche (P = 0.005). An OC patient with an age of menarche of 13 years or younger has 2.8 times the odds of having a family history of BC than those whose age of menarche is more than 13 years. Eleven percent of patients diagnosed with OC received no intervention. All other patients underwent treatment options including hysterectomy (69.5%), radiotherapy (39%) and chemotherapy (95%). The profiles of the patients showed that those who had a family history of OC were more likely to provide blood samples (OR = 3.87, P = 0.025), and similarly for those with a history of breast cancer (OR = 2.83, P = 0.022) in comparison to those who were not willing to provide blood for testing of biomarker. The distribution of CA125 for OC patients and control group showed that CA125 values were significantly higher (P = 0.034) in the case patients compared with the control group. The decrease in CA125 levels indicated the positive response to treatment, whereas increase in CA125 values showed resistant and disease progression. 52% of the patients with OC were correctly diagnosed as having OC (based on the optimal cut-point of CA125), while 83% of those without OC were also correctly diagnosed (with 48% of OC patients and 17% of non-OC patients incorrectly diagnosed). We concluded that there is significant association between family history of breast cancer and OC history and use of CA125 as a biomarker is not an ideal diagnostic and monitoring test as it has low sensitivity and high specificity.

Chemical-based Hydrogen Storage Systems: Recent Developments, Challenges, and Prospectives.

Hydrogen (H2) is being acknowledged as the future energy carrier due to its high energy density and potential to mitigate the intermittency of other renewable energy sources. H2 also ensures a clean, carbon-neutral, and sustainable environment for current and forthcoming generations by contributing to the global missions of decarbonization in the transportation, industrial, and building sectors. Several H2 storage technologies are available and have been employed for its secure and economical transport. The existing H2 storage and transportation technologies like liquid-state, cryogenic, or compressed hydrogen are in use but still suffer from significant challenges regarding successful realization at the commercial level. These factors affect the overall operational cost of technology. Therefore, H2 storage demands novel technologies that are safe for mobility, transportation, long-term storage, and yet it is cost-effective. This review article presents potential opportunities for H2 storage technologies, such as physical and chemical storage. The prime characteristics and requirements of H2 storage are briefly explained. A detailed discussion of chemical-based hydrogen storage systems such as metal hydrides, chemical hydrides (CH3OH, NH3, and HCOOH), and liquid organic hydrogen carriers (LOHCs) is presented. Furthermore, the recent developments and challenges regarding hydrogen storage, their real-world applications, and prospects have also been debated.

MXenes and MXene-Based Metal Hydrides for Solid-State Hydrogen Storage: A Review.

Hydrogen-driven energy is fascinating among the everlasting energy sources, particularly for stationary and onboard transportation applications. Efficient hydrogen storage presents a key challenge to accomplishing the sustainability goals of hydrogen economy. In this regard, solid-state hydrogen storage in nanomaterials, either physically or chemically adsorbed, has been considered a safe path to establishing sustainability goals. Though metal hydrides have been extensively explored, they fail to comply with the set targets for practical utilization. Recently, MXenes, both in bare form and hybrid state with metal hydrides, have proven their flair in ascertaining the hydrides' theoretical and experimental hydrogen storage capabilities far beyond the fancy materials and current state-of-the-art technologies. This review encompasses the significant accomplishments achieved by MXenes (primarily in 2019-2024) for enhancing the hydrogen storage performance of various metal hydride materials such as MgH2, AlH3, Mg(BH4)2, LiBH4, alanates, and composite hydrides. It also discusses the bottlenecks of metal hydrides for hydrogen storage, the potential use of MXenes hybrids, and their challenges, such as reversibility, H2 losses, slow kinetics, and thermodynamic barriers. Finally, it concludes with a detailed roadmap and recommendations for mechanistic-driven future studies propelling toward a breakthrough in solid material-driven hydrogen storage using cost-effective, efficient, and long-lasting solutions.

Exploring Nanomaterials for Hydrogen Storage: Advances, Challenges, and Perspectives.

Hydrogen energy heralded for its environmentally friendly, renewable, efficient, and cost-effective attributes, stands poised as the primary alternative to fossil fuels in the future. Despite its great potential, the low volumetric density presents a formidable challenge in hydrogen storage. Addressing this challenge necessitates exploring effective storage techniques for a sustainable hydrogen economy. Solid-state hydrogen storage in nanomaterials (physically or chemically) holds promise for achieving large-scale hydrogen storage applications. Such approaches offer benefits, including safety, compactness, lightness, reversibility, and efficient generation of pure hydrogen fuel under mild conditions. This article presents solid-state nanomaterials, specifically nanoporous carbons (activated carbon, carbon fibers), metal-organic frameworks, covalently connected frameworks, nanoporous organic polymers, and nanoscale metal hydrides. Furthermore, new developments in hydrogen fuel cell technology for stationary and mobile applications have been demonstrated. The review outlines significant advancements thus far, identifies key barriers to practical implementation, and presents a perspective for future sustainable energy research. It concludes with recommendations to enhance hydrogen storage performance for cost-effective and long-lasting utilization.

Welcoming the First Magnetic Resonance-Integrated Linear Accelerator in Pakistan.

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Facile hydrothermal synthesis of BiVO4 nanomaterials for degradation of industrial waste.

Bismuth Vanadate (BiVO4) has been synthesized using simple hydrothermal technique while varying the pH of concentrated H2SO4. With the increase of pH values (from 06 to 10), the morphology of the synthesized material tuned in the form of nano-spheres and cubes in the range from 50 to 60 nm. The lateral affect tuned the bandgap of BiVO4 from 2.47 eV to 2.50 eV which is significant in the context of present study. It is worth mentioning that desirous bandgap corresponds to the visible spectrum of the solar light being abundantly available and finds many applications in real life. The synthesized nanomaterial BiVO4 has been characterized through UV-Vis spectroscopy, X-ray diffraction, Scanning electron microscope and energy-dispersive X-ray (EDX) spectroscopy. The synthesized BiVO4 has been tested as photocatalyst for degradation of industrial pollutant from Leather Field Industry. Said catalyst (BiVO4) successfully degraded the industrial pollutant after 3 h under solar light irradiation. Therefore, the BiVO4 can be regarded as potential photocatalyst for degradation of industrial waste which is highly needed.

Radiotherapy and Limb-Sparing Surgery in the Management of Localized Soft Tissue Sarcomas: Tertiary Care Center Experience From Pakistan.

PURPOSE: When combined with radiotherapy, limb salvage surgery is an alternative to amputation. This study sought to determine the limb-sparing treatment outcomes in patients diagnosed with soft tissue extremity sarcomas treated at our institution. MATERIALS AND METHODS: All adult patients with extremity soft tissue sarcoma treated with the radical limb salvage strategy at Shaukat Khanum Memorial Cancer Hospital and Research Canter, Lahore, Pakistan, between January 2017 and December 2019 were retrospectively assessed. RESULTS: A total of 122 patients were included in the study. The mean age was 42 years (range 19-82), and 64 (52.5%) were males. The majority of patients, 65 (53.3%), were diagnosed with stage III and grade III disease according to American Joint Committee on Cancer TNM classification (Eighth edition). The most common surgical modality was wide local excision that was performed in 106 (86.9%) patients. Adjuvant radiation treatment was given in 111 (91%) patients, whereas 11 (9%) patients received neoadjuvant radiation treatment. The mean dose was 58 Gy (range: 46-66 Gy). Eighty-two (67.2%) of the patients were disease-free on post-treatment radiologic scans with disease recurrence observed in 40 (32.8%) patients. The median disease-free survival was 8 months (95% CI, 5.45 to 10.55). Local recurrence and distant metastases developed in 16 (13%) and 24 (20%) patients, respectively. CONCLUSION: About two thirds of patients with extremity soft tissue sarcoma were successfully treated with limb salvage strategy, surgery, and radiation therapy. However, high rate of relapse warrants further novel strategies in this patient population.

Expression and glucocorticoid-regulation of "Bam", a novel BH3-only transcript in acute lymphoblastic leukemia.

Apoptosis is a morphologically defined form of cell death that plays a major role in cell physiology, pathology and cancer therapy. The Bcl-2 family of pro- and anti-apoptotic molecules is a key regulator of this phenomenon, with the sub-family of BH3-only molecules serving as activators and/or facilitators. Apoptosis induced by glucocorticoids (GC) is a central component in the therapy of acute lymphoblastic leukemia (ALL), and defining its molecular basis and that of GC resistance is crucial for therapeutic improvements. We recently identified a novel transcript from the BCL2L11/Bim locus, termed "Bam", by affymetrix based whole genome expression profiling performed on 27 children (13 were already published) with ALL and many additional biological systems. Most children that induced Bam also induced Bim as well, in some cases Bam induction was more pronounced than that of Bim and in one patient only the Bam, but not Bim was induced. In C7H2, PreB697, Jurkat-GR(wt) and GC-sensitive (S-lines), this transcript was induced by GC in a translation-independent manner, suggesting that direct transcriptional induction of this BH3-only molecule by GC might cause apoptosis in at least ALL children and other biological systems.

Problems encountered in bicistronic IRES-GFP expression vectors employed in functional analyses of GC-induced genes.

Our laboratory has developed a series of Gateway(®) compatible lentiviral expression systems for constitutive and conditional gene knock-down and over-expression. For tetracycline-regulated transgenic expression, we constructed a lentiviral "DEST" plasmid (pHR-TetCMV-Dest-IRES-GFP5) containing a tetracycline-responsive minimal CMV promoter, followed by an attP site-flanked DEST cassette (for efficient cloning of cDNAs by "Gateway(®)" recombination cloning) and green fluorescent protein (GFP) driven by an internal ribosomal entry site (IRES).This lentiviral bicistronic plasmid allows immediate FACS identification and characterization of successfully transfected cell lines. Although this system worked well with several cDNAs, we experienced serious problems with SLA, Bam and BMF. Particularly, we cloned the cDNA for human SLA (Src-like adapter), a candidate gene in GC-induced apoptosis, into this plasmid. The resulting construct (pHR-TetCMV-SLA-IRES-GFP5) was transfected into HEK 293-T packaging cells to produce viral particles for transduction of CEM-C7H2-2C8 cells. Although the construct produced many green fluorescent colonies at the HEK 293-T and the CEM-C7H2-2C8 level, we could not detect any SLA protein with α-SLA antibody from corresponding cell lysates. In contrast, the antibody readily detected SLA in whole cell lysate of HEK 293-T cells transfected with a GST-flagged SLA construct lacking IRES-GFP. To directly address the potential role of the IRES-GFP sequence, we cloned the SLA coding region into pHR-TetCMV-Dest, a vector that differs from pHR-TetCMV-Dest-IRES-GFP5 just by the absence of the IRES-GFP cassette. The resulting pHR-TetCMV-SLA construct was used for transfection of HEK 293-T cells. Corresponding lysates were assayed with α-SLA antibody and found positive. These data, in concert with previous findings, suggest that the IRES-GFP cassette may interfere with translation of certain smaller size cDNAs (like SLA) or generate fusion proteins and entail defective virus production in an unpredictable manner.