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Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MAL TIR ). We show that o-vanillin binds to MAL TIR and inhibits its higher-order assembly in vitro . Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Water level fluctuations in China's Three Gorges Reservoir (TGR) area are typical of many reservoirs and significantly impact water level fluctuation zones (WLFZ), including upstream rivers. Understanding methane oxidation in the TGR-WLFZ is crucial for evaluating the impact of large-scale reservoir construction on global climate change. In this study, we investigated methane oxidation rates in the TGR-WLFZ, focusing on periods of drying and flooding. The highest methane oxidation rates were observed during the drying period, ranging from 35.69 to 56.32 nmol/(g soil)/d, while the lowest rates were recorded during the flooding period, at 11.58 to 11.98 nmol/(g soil)/d, in lab-scale simulated columns. Using 13 CH 4 labeling experiments, we measured CH 4 oxidation potentials for aerobic methane oxidation (AMO) using oxygen and anaerobic oxidation of methane (AOM) using nitrite, nitrate, sulfate, ferric iron, and manganese oxide as electron acceptors at varying concentrations. AMO was the dominant process across all experiments, with potentials ranging from 145.71 to 180.77 nmol 13 CO 2 /(g soil)/d. For AOM, metal-dependent oxidation, particularly with Fe (III) and Mn(IV), was predominant (12.64-17.59 and 3.91-12.69 nmol 13 CO 2 /(g soil)/d, respectively), followed by nitrite and nitrate-dependent pathways (1.49-9.10 nmol 13 CO 2 /(g soil)/d). Sulfate-dependent AOM was limited (1.33-3.27 nmol 13 CO 2 /(g soil)/d). Metagenomic analysis identified key microorganisms responsible for AMO, such as unclassified_f_Methylobacteriaeae and Methylobacterium sp., and for AOM are Ca. Methylomirabilis oxyfera, Ca. Methanoperedens nitroreducens and Ca. Methylomirabilis sp. Complete functional genes and enzymes for the methane oxidation and reverse methanogenesis pathways were obtained in each hydrological period, with the highest content during the drying period and the lowest during flooding. Our study shows that reservoirs, traditionally considered significant sources of methane, may also act as methane sinks. This finding raises new questions: How do different methane oxidation pathways respond to water level fluctuations in reservoirs, and are some pathways more resilient to changes in hydrological conditions?, Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Cyclin-dependent kinase 9 (CDK9) stimulates oncogenic transcriptional pathways in cancer and CDK9 inhibitors have emerged as promising therapeutic candidates., Methods: The activity of an orally bioavailable CDK9 inhibitor, CDKI-73, was evaluated in prostate cancer cell lines, a xenograft mouse model, and patient-derived tumor explants and organoids. Expression of CDK9 was evaluated in clinical specimens by mining public datasets and immunohistochemistry. Effects of CDKI-73 on prostate cancer cells were determined by cell-based assays, molecular profiling and transcriptomic/epigenomic approaches., Results: CDKI-73 inhibited proliferation and enhanced cell death in diverse in vitro and in vivo models of androgen receptor (AR)-driven and AR-independent models. Mechanistically, CDKI-73-mediated inhibition of RNA polymerase II serine 2 phosphorylation resulted in reduced expression of BCL-2 anti-apoptotic factors and transcriptional defects. Transcriptomic and epigenomic approaches revealed that CDKI-73 suppressed signaling pathways regulated by AR, MYC, and BRD4, key drivers of dysregulated transcription in prostate cancer, and reprogrammed cancer-associated super-enhancers. These latter findings prompted the evaluation of CDKI-73 with the BRD4 inhibitor AZD5153, a combination that was synergistic in patient-derived organoids and in vivo., Conclusion: Our work demonstrates that CDK9 inhibition disrupts multiple oncogenic pathways and positions CDKI-73 as a promising therapeutic agent for prostate cancer, particularly aggressive, therapy-resistant subtypes., (© 2024. The Author(s).)

Metal pollution in sediment from construction areas raises ecological and health concerns, yet source-based sediment pollution in Bangladesh remains understudied. Our investigation focused on fifteen locations in the Kohelia River and the coastal regions near the Matarbari projects (Matarbari Power Plant, Matarbari Deep Seaport), assessing metal concentrations' sources and impacts on ecology and human well-being. Sediment quality indices indicated high Cd and Cr contamination, with sites near Matarbari projects being the most polluted. The positive matrix factorization model identified three anthropogenic sources and mixed sources. Matarbari projects contributed significantly to As (67.9 %), Mn (50.25 %), Cd (48.35 %), and Cr (41.0 %), while ship-breaking yards contributed Fe (58.0 %), Zn (55.5 %), Pb (53.8 %), and Cu (36.1 %). Ecological indices showed different impacts on aquatic life from metal pollution, but cancer risk levels stayed below the threshold set by the US Environmental Protection Agency. These findings underscore the need for targeted measures to address metal pollution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The organic matter (OM) and nitrogen in Fresh leachate (FL) from waste compression sites pose environmental and health risks. Even though the constructed wetland (CW) can efficiently remove these pollutants, the molecular-level transformations of dissolved OM (DOM) in FL remain uncertain. This study reports the molecular dynamics of DOM and nitrogen removal during FL treatment in CWs. Two lab-scale vertical-flow CW systems were employed: one using only sand as substrates (act as a control, CW-C) and the other employing an equal mixture of manganese ore powder and sand (experimental, CW-M). Over 488 days of operation, CW-M exhibited significantly higher removal rates for chemical oxygen demand (COD), ammonia nitrogen (NH 4 + -N), and dissolved organic matter (represented by dissolved organic carbon, DOC) at 98.2 ± 2.5%, 99.2 ± 1.4%, and 97.9 ± 1.9%, respectively, in contrast to CW-C (92.8 ± 6.8%, 77.1 ± 28.1%, and 74.7 ± 9.5%). The three-dimensional fluorescence excitation-emission matrix (3D-EEM) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analyses unveiled that the influent DOM was predominantly composed of readily biodegradable protein-like substances with high carbon content and low unsaturation. Throughout treatment, it led to the degradation of low O/C and high H/C compounds, resulting in the formation of DOM with higher unsaturation and aromaticity, resembling humic-like substances. CW-M showcased a distinct DOM composition, characterized by lower carbon content yet higher unsaturation and aromaticity than CW-C. The study also identified the presence of Gammaproteobacteria, reported as Mn-oxidizing bacteria with significantly higher abundance in the upper and middle layers of CW-M, facilitating manganese cycling and improving DOM removal. Key pathways contributing to DOM removal encompassed adsorption, catalytic oxidation by manganese oxides, and microbial degradation. This study offers novel insights into DOM transformation and removal from FL during CW treatment, which will facilitate better design and enhanced performance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jun Zhai reports financial support was provided by National Natural Science Foundation of China. Jun Zhai reports financial support was provided by Chongqing Science and Technology Project. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Periodic and significant water level fluctuations within the Three Gorges Reservoir (TGR) create a complex water level fluctuation zone (WLFZ) that can significantly influence greenhouse gas emissions. However, the scarcity of comprehensive studies investigating long-term monitoring and analysis of CH 4 flux patterns and underlying mechanisms concerning water level variations, environmental characteristics, and microbial communities has limited our understanding. This study conducted a four-year monitoring campaign to examine in situ CH 4 emissions from three representative sampling sites. Results indicated that the CH 4 flux remained relatively stable at lower water levels, specifically at the control site (S1). However, water level fluctuations significantly influenced CH 4 emissions at the sampling sites situated within the WLFZ. Notably, the highest CH 4 flux of 0.252 ± 0.089 mg/(m 2 ·h) was observed during the drying period (June to August), while the lowest CH 4 flux of 0.048 ± 0.026 mg/(m 2 ·h) was recorded during the flooding period. Moreover, CH 4 emissions through the water-air interface surpassed those through the soil-air interface. The CH 4 flux positively correlated with organic carbon, temperature, and soil moisture. The relative abundance of methane metabolism microorganisms peaked during the drying period and decreased during the impounding and flooding periods. The primary methanogenesis pathway was hydrogenotrophic, whereas methanotrophic processes were mainly aerobic, with Ca. Methylomirabilis governing the anaerobic methanotrophic process. Overall, the current findings serve as crucial theoretical references for understanding CH 4 emissions and carbon metabolism processes within WLFZ environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study attempted to develop a low-cost and eco-friendly bio-based composite adsorbent that is highly efficient in capturing potential toxic metals. The bio-composite adsorbent was prepared using graphene oxide (GO), carboxymethyl cellulose (CMC) and chitosan (CS); and characterized using FTIR, SEM-EDX and WAXD techniques. Metal-ion concentration in an aqueous solution was measured by ICP-OES. This article reveals that the adsorption of heavy metal ions varied according to the adsorbent quantity, initial metal concentration, pH, and interaction time. The metal ions' adsorption capacity (mg/g) was observed to increase when the interaction time and metal concentration increased. Conversely, metal ions adsorption was decreased with an increase in adsorbent dosages. The effect of pH on metal ions' adsorption was ion-specific. The substantial adsorption by GO/CMC/CS composite for Co 2+ , CrO 4 2- , Mn 2+ and Cd 2+ , had the respective values of 43.55, 77.70, 57.78, and 91.38 mg/g under acidic conditions. The metal ions experimental data were best fitted with pseudo-second-order (PSO) kinetics, and Freundlich isotherm model (except Co 2+ ). The separation factors (R L ) value in the present investigation were found between 0 and 1, meaning that the metal ions adsorption onto GO/CS/CMC composite is favorable. The R L and sorption intensity (1/n) values fitted well to the adsorption isotherm., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Silicon T centers present the promising possibility of generating optically active spin qubits in an all-silicon device. However, these color centers exhibit long excited state lifetimes and a low Debye-Waller factor, making them dim emitters with low efficiency into the zero-phonon line. Nanophotonic cavities can solve this problem by enhancing radiative emission into the zero-phonon line through the Purcell effect. In this work, we demonstrate cavity-enhanced emission from a single T center in a nanophotonic cavity. We achieve a 2 order of magnitude increase in the brightness of the zero-phonon line relative to waveguide-coupled emitters, a 23% collection efficiency from emitter to fiber, and an overall emission efficiency into the zero-phonon line of 63.4%. We also observe a lifetime enhancement of 5, corresponding to a Purcell factor exceeding 18 when correcting for the emission to the phonon sideband. These results pave the way toward efficient spin-photon interfaces in silicon photonics.

Microplastics (MPs) are ubiquitous pollutants that significantly threaten organisms and ecosystems. Constructed wetlands (CWs), a nature-based treatment technology, can effectively remove MPs from wastewater. However, the responses of CWs when exposed to MPs remain unclear. In this study, lab-scale vertical flow constructed wetlands (VFCWs) were installed for receiving polystyrene (PS) MPs at concentrations of 100 μg/L and 1000 μg/L. The results showed that exposure to PS-MPs has no effects on COD and TP removal in VFCWs, but TN removal decreased by 3.69-5.37 %. Further investigation revealed that PS-MPs significantly impacted microbial communities and metabolic functions. The abundances of predominant nitrifiers (Nitrospira and Nitrosomonas) and denitrifiers (Nakamurella, Bradyrhizobium, and Bacillus) in VFCWs were significantly reduced, aligning with the responses of key enzymes. The presence of PS-MPs also decreased nitrogen removal by plant uptake, leading to decreased plant biomass and chlorophyll by 39.32-48.75 % and 5.92-32.19 %, respectively. Notably, > 90 % removal rates were observed for PS-MPs within VFCWs. In addition to PS-MPs interception by VFCWs substrate, the increase of released benzenes indicated that the PS-MPs biodegradation occurred. Such insights are vital for developing sustainable solutions to mitigate MPs' adverse effects on ecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Soil erosion is the inherent and destructive threat affecting agricultural production and livelihood of million mouths. The increased frequency of floods and land use/land cover changes has made Upper Jhelum Sub-catchment susceptible to soil erosion risk. Morphometric based watershed prioritization for soil erosion risk may help in sustainable management of natural resources. Thus, this paper endeavors to prioritize watersheds of Upper Jhelum Sub-catchment in India based on morphometric parameters for soil erosion risk using geospatial techniques. Weights to the morphometric parameters were assigned through a multi-criteria decision method. The watersheds in the Sub-catchment have been categorized into low, medium, high and very high priority classes based on prioritization ranks that were determined by computing the compound value for the soil erosion risk, based on prioritization ranks obtained through compound value for the soil erosion risk. The results revealed 1E1D3 and 1E1D8 watersheds accorded very high priority. The watersheds namely IE1D2 and IEID4 were found under high priority. Medium priority for soil erosion risk was determined in IEID5 and IED7 watersheds while 1E1D1 and IE1D6 watersheds were identified for low priority. The study calls for implementing soil conservation practices in the Sub-catchment. The Sub-catchment can be made less hazardous for the soil erosion risk by implementing contour farming, building check dams, terrace farming, afforestation and limiting large scale overgrazing. The findings of this study may offer valuable insights for stakeholders for conservation of soil resource. The approach utilized in the study may be linked with soil loss estimation for effective conservation of natural resources in further future studies., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The heterogeneous integration of silicon with III-V materials provides a way to overcome silicon's limited optical properties toward a broad range of photonic applications. Hybrid modes are a promising way to integrate such heterogeneous Si/III-V devices, but it remains unclear how to utilize these modes to achieve photonic crystal cavities. Herein, using 3D finite-difference time-domain simulations, we propose a hybrid Si-GaAs photonic crystal cavity design that operates at telecom wavelengths and can be fabricated without requiring careful alignment. The hybrid cavity consists of a patterned silicon waveguide that is coupled to a wider GaAs slab featuring InAs quantum dots. We show that by changing the width of the silicon cavity waveguide, we can engineer the hybrid modes and control the degree of coupling to the active material in the GaAs slab. This provides the ability to tune the cavity quality factor while balancing the device's optical gain and nonlinearity. With this design, we demonstrate cavity mode confinement in the GaAs slab without directly patterning it, enabling strong interaction with the embedded quantum dots for applications such as low-power-threshold lasing and optical bistability (156 nW and 18.1 µW, respectively). This heterogeneous integration of an active III-V material with silicon via a hybrid cavity design suggests a promising approach for achieving on-chip light generation and low-power nonlinear platforms.

Acute myeloid leukaemia (AML) affects predominantly elderly people and has an incidence of 1% of all cancers and 2% of all cancer deaths. Despite using intensive chemotherapy and allogeneic stem cell transplantation, the treatment options for AML remain open for innovation. Thus, there is a need to explore alternative therapies such as less toxic targeted therapies in AML. Aurora A kinase is a well-established target for the treatment of various cancers, including AML. This kinase plays a pivotal role in the cell-division cycle, particularly in different stages of mitosis, and is also involved in many other cellular regulatory processes. In a previous study, we demonstrated that the anti-viral drug rilpivirine is an Aurora A kinase inhibitor. In the current study, we have further explored the selectivity of rilpivirine for Aurora A kinase inhibition by testing this drug against a panel of 429 kinases. Concurrently, we demonstrated that rilpivirine significantly inhibited the proliferation of AML cells in a time- and concentration-dependent manner that was preceded by G 2 /M cell-cycle arrest leading to the induction of apoptosis. Consistent with its kinase inhibitory role, rilpivirine modulated the expression of critical proteins in the Aurora A kinase-signalling pathway. Importantly, orally administered rilpivirine significantly inhibited tumour growth in an HL-60 xenograft model without showing body weight changes or other clinical signs of toxicity. Furthermore, rilpivirine enhanced the anti-proliferative efficacy of the conventional anti-leukaemic chemotherapeutic agent cytarabine. Collectively, these findings provide the stimulus to explore further the anti-leukaemic activity of the anti-viral drug rilpivirine.

The main objective of this study is to assess the impacts of cooking and indoor air quality (IAQ) in the southwestern region of Bangladesh. Here we report and compare the IAQ in considering a total of eight kitchens and living rooms of four selected households (HHs) in Jashore city and suburb area, the southwest district of Bangladesh. Air quality parameters, such as particulate matter (PM 2.5 ) and volatile organic compounds (VOC), were assessed continuously for 24 h. In addition, Carbon dioxide (CO 2 ) was evaluated in different phases during the study period. PM 2.5 , VOC, and CO 2 levels were ranged from 18.52 to 207 μg/m 3 , 7.95-35.66 ppm, and 1061-2459 mg/m 3 , respectively, in the indoor cooking HHs. Conversely, while the average concentration was found between 20.63 and 23.72 μg/m 3 PM 2.5 , 11.18-12.36 ppm VOC, and 1097-1747 mg/m 3 CO 2 in the outdoor cooking HHs. A significant increase in CO 2 due to kitchen activities (cooking, frying, boiling) was observed that ranged between 5 and 77% compared to the background level. The calculated range of toxicity potential (TP) values was between 0.8 and 8.3 for PM 2.5 in the HHs. In most of the observations, PM 2.5 , VOC, and CO 2 exceed the standard values. The study reports that well ventilation systems and clean fuel use significantly reduce the indoor air contaminants level. Our study offers new insights about the IAQ of the southwest region of Bangladesh, particularly for suburbs and urban setups, and provides a background for further study, and decision-making. It will serve as a reference for the formulation and implementation of policies to improve air quality., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors.)

Globally, floods as dynamic hydraulic hazard have caused widespread damages to both socioeconomic conditions and environment at various scales. Managing flood and management of water resource is a global challenge under the changing climatic condition. This study assessed flood susceptibility in the Bhagirathi sub-basin, India using entropy information theory and geospatial technology. Twelve flood susceptibility parameters such as land use/land cover, normalized difference vegetation index (NDVI), slope, elevation, geology, geomorphology, normalized difference water index (NDWI), soil, drainage density, average rainfall, maximum temperature, and humidity during monsoon season were utilized to examine flood susceptibility. Receiver operating characteristics (ROC) curve and Leave-One-Out Cross-Validation (LOOCV) techniques were carried out to validate flood susceptibility map. Kappa statistics was also used to check the reliability of the flood susceptibility model. Findings of the study revealed that nearly 45% area of the sub-basin was highly susceptible to flood followed by moderate (29.3%), very high (19%), low (6.9%), and very low (0.2%). These findings also revealed that nearly 92% area in the eastern, north-eastern, and deltaic sub-basin was susceptible to floods. ROC analysis indicated high success (0.932) and prediction (0.903) rates for the susceptibility map while LOOCV (R 2 being 0.97) and Kappa (k = 0.934) have shown substantial prediction of the model. Hence, the susceptibility maps are useful for the local planners and government organization in designing the early flood warning system, and reducing the human and economic losses. The methodology used in this study is applicable for analyzing flood susceptibility at spatial scales in similar systems., (© 2022 Society for Risk Analysis.)

Multivariate statistics and GIS alone with geochemical modeling were applied to investigate the hydro-geochemical characteristics of groundwater and their spatial distribution in the deep aquifer system of Bagherpara Upazila, Bangladesh. This Upazila consists of an area of 308.3 km 2 and local people mostly rely on groundwater to meet the drinking water requirements. Water samples from one hundred randomly selected deep tube wells (152-198 m) were collected and analyzed for 14 water quality parameters to characterize the hydro-geochemical properties. The groundwater shows slight alkaline in nature throughout the study area. Most of the water samples were turbid and 68% of them failed to meet the drinking water quality standard prescribed by the World Health Organization (WHO). TDS concentration ranges between 280 mg/L and 1040 mg/L, with a mean value of 446.20 (±122.19) mg/L. The general order of cation and anion along the study area were Ca 2+ >Mg 2+ >Na + >K + and HCO 3 - >Cl - >PO 4 3- >SO 4 2- >NO 3 - respectively. Carbonate weathering is the dominant process for releasing ions in groundwater. Besides, the ion exchange process is active in the study area, which leads to the reduction of Na + ions. Gibb's plot suggests a rock dominance inheritance controls the dissolution and precipitation of minerals along with the ion-exchange process, and ultimately dictates the groundwater chemistry. Besides, the Piper diagram showed that Ca 2+ -Mg 2+ -SO 4 2- is the dominant water type in 65% of the samples followed by Ca 2+ -Mg 2+ -HCO 3 - water type (35%). The abundance of Ca 2+ and Mg 2+ ions and the alkaline nature of groundwater indicate mixed geochemical facies and reverse exchange reactions. The principal component analysis (PCA) reveals that weathering and leaching of host rocks was the main natural source, while municipal solid waste dumping, sewage discharge, and fertilizer application could be other anthropogenic factors that affect the groundwater geochemistry. In fine, the chemical characteristics of groundwater were acquired through rock dissolution, percolation, and reverse exchange process., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Salinity intrusion both in surface and groundwater caused a crisis for safe drinking water in coastal Bangladesh. The situation is even worse for children especially at school. However, information on water services in coastal schools is limited. Here we assess the quality of drinking water and supply infrastructures in the primary schools of a severely saline affected coastal area of Bangladesh. To fulfill the objective, thirty-eight schools were purposively selected and investigated in Dacope Upazila of Khulna district in Bangladesh. Findings revealed that harvested rainwater (63%) and pond (21%) are the major drinking water sources where countries' leading water supply technology, tube well (16%) were the least used option. Moreover, salinity in all the tube wells exceeded the national standard. DO, pH, NO 3 , SO 4 and PO 4 concentration of all options satisfied national standards. However, total coliform counts exceeded the national standard. More than half of the samples had a low to high risk of indicator bacteria which is a major public health concern. Although 29% schools have installed portable water filtration units, those are grossly inaccessible for the students. Hence, students are reportedly consuming unsafe drinking water, and thus are vulnerable to water-borne diseases. The lack of resources and poorly designed infrastructure are the principal challenges to the safe drinking water supply. Therefore, disinfection at the point of use along with proper maintenance of the water infrastructure is urgent needs to safeguard potable water services in the primary schools of coastal Bangladesh., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Soil salinity in the coastal areas of Bangladesh has been rising. The responses of forest communities to the rise of salinity are somehow documented. However, the adjustments of non-forest plant communities against salinity are still limited in the literature. This study explains the phytosociology and the herbaceous plant biodiversity along with the soil salinity gradients in Shyamnagar, Bangladesh. Twenty-five roadside quadrants were systematically selected and studied for herbaceous plant diversity and soil properties. Soil Electrical conductivity (EC) and moisture show a generally rising trend from the north to the south. Moreover, the quadrants closer to the river or aquaculture with low elevation represent the salinity hot spots. About 1116 herbaceous plants belonging to 11 species were recorded. Croton bonplandianum baill is the dominant species and showed higher adaption capacity against soil EC values. Four out of 25 quadrants with higher EC, moisture and lower elevation recorded no plants. The herbaceous plant biodiversity reveals a strong spatial pattern and tendency to shrink with the rise of soil salinity while progressing towards the southeast direction. The area shows aggregated population, contagious distribution of plant species, and accommodates four dominant clusters. Roadside herbs not only hold medicinal value but also offer important cooking fuel choices to the fuel-scarce coastal communities. The outcome of this study provides important insights into herbaceous plant diversity and its relationships with soil salinity. Overall, the study outcomes improve our understanding of the impact of environmental stressors on the distribution of herbaceous plants in the coastal area., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

The coupling transformation of carbon, nitrogen and sulfur compounds has been studied in lab-scale and pilot-scale constructed wetlands (CWs), but few studies investigated full-scale CW. In this study, we used batch experiments to investigate the potentials of carbon, nitrogen and sulfur transformation in a long-term operated, full-scale horizontal subsurface flow wetland. The sediments collected from the HSFW were incubated for 48 h in the laboratory with supplying various dosages of carbon, nitrogen and sulfur compounds. The results showed that heterotrophic denitrification was the main pathway. At the same time, the sulfide (S 2- )-based autotrophic denitrification was also present. Increasing TOC concentration or NO 3 - concentration could promote heterotrophic denitrification but did not inhibit the sulfide-based autotrophic denitrification. In our experiment, the highest NO 3 - removal via autotrophic denitrification was 25.23% while that via heterotrophic denitrification was 73.66%, leading to the total NO 3 - removal of 98.89%. The results also demonstrated that NO 3 - rather than NO 2 - was the preferable electron acceptor for both heterotrophic and sulfide-based autotrophic denitrification in the CW. Increasing S 2- concentrations promote NO 3 - removal from 12.99% to 25.23% without organic carbon, but varying NO 3 - or NO 2 - has no effects. These results indicated that concentrations of S 2- , instead of NO 3 - or NO 2 - , was the limiting factor for sulfide-based autotrophic denitrification in the studied CW. The microbial community analysis and correlation analysis between the transformation of carbon, nitrogen and sulfur compounds and relative abundance of bacteria further confirmed that in the CW, the key pathways coupling transformation were heterotrophic denitrification and sulfide-based autotrophic denitrification. Overall, the current study will enhance understanding of carbon, nitrogen, and sulfur transformation in CW and support better design and treatment efficiency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Organotrophic anammox is a promising process for treating both nitrogen and organic containing wastewater than that of the traditional autotrophic anammox for sole nitrogen removal. However pathways of nitrogen removal particularly at metagenomic level in both processes are still unknown. Here we report, metabolic pathways of nitrogen removal in two lab-scale sequencing batch reactors (SBR), one autotrophic and another organotrophic (TOC/TN = 0.1) anammox bacteria incubated over 220 days. Both reactors showed satisfactory nitrogen removal with 840.31 mg N/L.d and 786.81 mg N/L.d for autotrophic and organotrophic anammox reactors respectively. Four anammox species namely Candidatus B. fulgida, B. sinica, J. caeni and Candidatus K. stuttgartiensis were identified in both reactors. The Candidatus K. stuttgartiensis (4%) was dominant in autotrophic reactor whereas Candidatus J. caeni (10%) in the organotrophic reactor. The supply of organic promoted the growth of anammox bacteria more than three times higher than that of the autotrophic anammox reactor. The functional genes related to the DNRA pathway was obtained in all anammox species except for Candidatus K. stuttgartiensis. The co-existence of other DNRA (Armatimonadetes and Thauera) and partial denitrifying bacteria (Chloroflexi) was also found in both reactors. Moreover, functional genes related to acetate metabolism by acetyl-CoA way were obtained in all anammox bacteria except Candidatus B. fulgida which showed alternative ackA/Pac-t pathways in organic anammox reactor. Overall current results suggest that the anammox, DNRA and partial denitrification were the key nitrogen transformation pathways, particularly in organotrophic anammox reactor. Our findings will improve understanding of the practical application of organotrophic anammox for wider wastewater treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL TIR ) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88 TIR ). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88 TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MAL TIR . We demonstrate via mutagenesis that the MyD88 TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88 TIR . Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

CDK8 regulates transcription either by phosphorylation of transcription factors or, as part of a four-subunit kinase module, through a reversible association of the kinase module with the Mediator complex, a highly conserved transcriptional coactivator. Deregulation of CDK8 has been found in various types of human cancer, while the role of CDK8 in supressing anti-cancer response of natural killer cells is being understood. Currently, CDK8-targeting cancer drugs are highly sought-after. Herein we detail the discovery of a series of novel pyridine-derived CDK8 inhibitors. Medicinal chemistry optimisation gave rise to 38 (AU1-100), a potent CDK8 inhibitor with oral bioavailability. The compound inhibited the proliferation of MV4-11 acute myeloid leukaemia cells with the kinase activity of cellular CDK8 dampened. No systemic toxicology was observed in the mice treated with 38. These results warrant further pre-clinical studies of 38 as an anti-cancer agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88. Using targeted mutagenesis and protein-protein crosslinking, we show that A46 interacts with MAL and MyD88 through several facets, including residues on helices α1 and α7 and the C-terminal flexible region. We propose a model in which A46 targets the MAL and MyD88 signalosome intra-strand interfaces and gradually destroys their assemblies in a concentration-dependent manner., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cyclin-dependent kinase (CDK) 12 engages in diversified biological functions, from transcription, post-transcriptional modification, cell cycle, and translation to cellular proliferation. Moreover, it regulates the expression of cancer-related genes involved in DNA damage response (DDR) and replication, which are responsible for maintaining genomic stability. CDK12 emerges as an oncogene or tumor suppressor in different cellular contexts, where its dysregulation results in tumorigenesis. Current CDK12 inhibitors are nonselective, which impedes the process of pharmacological target validation and drug development. Herein, we discuss the latest understanding of the biological roles of CDK12 in cancers and provide molecular analyses of CDK12 inhibitors to guide the rational design of selective inhibitors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Innate immunity pathways constitute the first line of defense against infections and cellular damage. An emerging concept in these pathways is that signaling involves the formation of finite (e.g. rings in NLRs) or open-ended higher-order assemblies (e.g. filamentous assemblies by members of the death-fold family and TIR domains). This signaling by cooperative assembly formation (SCAF) mechanism allows rapid and strongly amplified responses to minute amounts of stimulus. While the characterization of the molecular mechanisms of SCAF has seen rapid progress, little is known about its regulation. One emerging theme involves proteins produced both in host cells and by pathogens that appear to mimic the signaling components. Recently characterized examples involve the capping of the filamentous assemblies formed by caspase-1 CARDs by the CARD-only protein INCA, and those formed by caspase-8 by the DED-containing protein MC159. By contrast, the CARD-only protein ICEBERG and the DED-containing protein cFLIP incorporate into signaling filaments and presumably interfere with proximity based activation of caspases. We review selected examples of SCAF in innate immunity pathways and focus on the current knowledge on signaling component mimics produced by mammalian and pathogen cells and what is known about their mechanisms of action., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Colorectal cancer (CRC) remains one of the most lethal human malignancies, and pursuit of new therapeutic targets for treatment has been a major research focus. Cyclin-dependent kinase 9 (CDK9), which plays a crucial role in transcription, has emerged as a target for cancer treatment. CDKI-73, one of the most potent and pharmacologically superior CDK9 inhibitors, has demonstrated excellent anti-tumour efficacy against several types of cancers. In this study, we evaluated its therapeutic potential against CRC. CDKI-73 elicited high cytotoxicity against all colon cancer cell lines tested. Cell cycle and apoptosis analysis in HCT 116 and HT29 cells revealed that CDKI-73 induced cell death without accumulation of DNA at any phase of the cell cycle. Moreover, it caused depolarisation of mitochondrial membrane, leading to caspase-independent apoptosis. Knockdown by shRNA demonstrated the CDK9-targeted mechanism of CDKI-73, which also affected the Mnk/eIF4E signalling axis. In addition, RT-qPCR analysis showed that CDKI-73 down-regulated multiple pro-survival factors at the mRNA level. Its in vivo anti-tumour efficacy was further evaluated in Balb/c nude mice bearing HCT 116 xenograft tumours. CDKI-73 significantly inhibited tumour growth (***P < 0.001) without overt toxicity. Analysis of the tumour tissues collected from the xenografted animals confirmed that the in vivo anti-tumour efficacy was associated with CDK9 targeting of CDKI-73. Overall, this study provides compelling evidence that CDKI-73 is a promising drug candidate for treating colorectal cancer., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Acute myeloid leukemia (AML) is the most common form of acute leukemia with dismal long-term prognosis with age. The most aggressive subtype of AML is MLL-AML that is characterized by translocations of the mixed-lineage leukemia gene (MLL) and resistance to conventional chemotherapy. Cyclin dependent kinase 9 (CDK9) plays a crucial role in the MLL-driven oncogenic transcription, and hence, inhibiting activity of CDK9 has been proposed as a promising strategy for treatment of AML. We investigated the therapeutic potential of CDKI-73, one of the most potent CDK9 inhibitors, against a panel of AML cell lines and samples derived from 97 patients. CDKI-73 induced cancer cells undergoing apoptosis through transcriptional downregulation of anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP by majorly targeting CDK9. Contrastively, it was relatively low toxic to the bone marrow cells of healthy donors. In MV4-11 xenograft mouse models, oral administration of CDKI-73 resulted in a marked inhibition of tumor growth (p < 0.0001) and prolongation of animal life span (P < 0.001) without causing body weight loss and other overt toxicities. The study suggests that CDKI-73 can be developed as a highly efficacious and orally deliverable therapeutic agent for treatment of AML.

This study reports the fast hydrogenation and dehydrogenation of ultra-thin discrete platinum/palladium (Pt/Pd) bimetal over nano-structured Ag islands grown on rough alumina substrate by a RF magnetron sputtering technique. The morphology of Ag nanoislands was optimized by RF magnetron sputtering and rapid thermal annealing process. Later, Pt/Pd bimetal (10/10) nm were deposited by RF magnetron sputtering on the nanostructured Ag islands. After the surface morphological optimization of Ag nanoislands, the resultant structure Pt/Pd@Ag nanoislands at alumina substrate showed a fast and enhanced hydrogenation and dehydrogenation (20/25 s), response magnitude of 2.3% (10,000 ppm), and a broad detection range of 500 to 40,000 ppm at the operating temperature of 120 °C. The superior hydrogenation and dehydrogenation features can be attributed to the hydrogen induced changes in the work function of Pt/Pd bimetal which enhances the coulomb scattering of percolated Pt/Pd@Ag nanoislands. More importantly, the atomic arrangements and synergetic effects of complex metal alloy interfacial structure on Ag nanoislands, supported by rough alumina substrate incorporate the vital role in accelerating the H₂ absorption and desorption properties.

Carbamazepine (CBZ), a widely used antiepileptic drug, is refractory to biological wastewater treatment. Rapid removal of CBZ is possible using synthetic manganese oxide (δ-MnO 2 ) but the removal mechanisms require further investigation. In this study, CBZ degradation by δ-MnO 2 was carried out at different pH to further explore the degradation mechanisms. Results show that CBZ degradation by δ-MnO 2 was highly pH dependent, and rapid degradation occurred when pH <2.8. Based on the density functional theory calculations, increasing [H + ] not only increased the reactivity of δ-MnO 2 , but also enhanced the secondary reactions of the intermediates. During the degradation process, protonation of CBZ degradation intermediates, instead of CBZ, played an important role. The overall kinetics of CBZ degradation was then described by the retarded first-order model. The initial rate (r init ) in the model between pH 2.0 and 6.2 was determined to be r init  = (2.41 ± 0.51) × 10 -3 [CBZ] 1.21 [MnO 2 ] 1.07 [H + ] 1.41 . This is the first report revealing that protonation of intermediates from CBZ degradation can improve the CBZ oxidation by δ-MnO2. The pathways of CBZ degradation by δ-MnO 2 were also proposed. The results of this study provide a new insight into the processing mechanism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The discovery of novel anti-AML therapeutic agents is urgently needed, but the complex heterogeneity of the disease has so far hampered the development of a curative treatment. FLT3 inhibitors have shown therapeutic potential in clinical trials; but a monotherapy regimen has been associated with resistance mediated by the activation of parallel signalling circuitry, including MAPK and mTOR. Therefore, inhibiting a nexus of the two signalling pathways along with inhibition of FLT3 might be advantageous. Herein, we propose that a dual inhibition of FLT3 and Mnk would provide a better clinical option for AML patients compared to targeting FLT3 alone. Thus, a series of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines and 4-(indol-3-yl)-N-phenylpyrimidin-2-amines were prepared. Potent Mnk2 inhibitors, FLT3 inhibitors, and dual inhibitors of Mnk2 and FLT3 were identified and their anti-proliferative activities assessed against MV4-11 AML cell lines. Dual inhibition of FLT3 and Mnk2 caused the increased apoptotic cell death of MV4-11 cells compared to inhibition of FLT3 or Mnk2 alone., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

This study aimed to optimise coagulation pretreatment of the produced water (PW) collected from a natural gas field. Two coagulants, polyferric sulphate (PFS) and polyaluminium chloride (PACl), were applied separately for the organics, suspended solids (SS), and colour removal. Treatment performance at different coagulant dosages, initial pH values, stirring patterns, and the addition of cationic polyacrylamide (PAM) was investigated in jar tests. The optimal coagulation conditions were dosage of PACl 25 g/L or PFS 20 g/L with that of PAM 30 mg/L, initial pH of 11, and fast mixing of 1.5 min (for PACl) or 2 min (for PFS) at 250 rpm followed by slow mixing of 15 min at 50 rpm for both coagulants. PACl performed better than PFS to remove chemical oxygen demand (COD), total organic carbon (TOC), SS, and colour, and achieved a removal efficiency of 90.1%, 89.4%, 99.0%, and 99.9%, respectively, under the optimal condition; while PFS efficiency was 86.1%, 86.1%, 99.0%, and 98.2%, respectively. However, oil removal was higher in PFS coagulation compared to PACl and showed 98.9% and 95.3%, respectively. Biodegradability, ratio of the biological oxygen demand (five-day) (BOD 5 )/COD, of the PW after pretreatment increased from 0.08 to 0.32 for PFS and 0.43 for PACl. Zeta potential (Z-potential) analysis at the optimum coagulant dosage of PACl and PFS suggests that charge neutralisation was the predominant mechanism during coagulation. Better efficiency was observed at higher pH. The addition of PAM and starring pattern had a minor influence on the removal performance of both coagulants. The results suggest that PACl or PFS can be applied for the pretreatment of PW, which can provide substantial removal of carbon, oil, and colour, a necessary first step for subsequent main treatment units such as chemical oxidation or biological treatment.

Cyclin D dependent kinases (CDK4 and CDK6) regulate entry into S phase of the cell cycle and are validated targets for anticancer drug discovery. Herein we detail the discovery of a novel series of 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine derivatives as highly potent and selective inhibitors of CDK4 and CDK6. Medicinal chemistry optimization resulted in 83, an orally bioavailable inhibitor molecule with remarkable selectivity. Repeated oral administration of 83 caused marked inhibition of tumor growth in MV4-11 acute myeloid leukemia mouse xenografts without having a negative effect on body weight and showing any sign of clinical toxicity. The data merit 83 as a clinical development candidate.

Cyclin-dependent kinase 9 (CDK9) is a key transcriptional regulator and a lucrative target for cancer treatment. Targeting CDK9 can effectively confine the hyperactivity of androgen receptor and the constitutive expression of anti-apoptotic proteins; both being main causes of prostate cancer (PCa) development and progression. In castrate-resistant PCa, traditional therapies that only target androgen receptor (AR) have become obsolete due to reprograming in AR activity to make the cells independent of androgen. CDK9 inhibitors may provide a new and better therapeutic opportunity over traditional treatment options by targeting both androgen receptor activity and anti-apoptotic proteins, improving the chances of positive outcomes, especially in patients with the advanced disease. This review focuses on biological functions of CDK9, its involvement with AR and the potential for therapeutic opportunities in PCa treatment., (© 2016 Society for Endocrinology.)

This study aimed to assess the uptake of diclofenac, a widely used nonsteroidal anti-inflammatory pharmaceutical, by a macrophyte Cyperus alternifolius in a mesocosm-scale free water surface (FWS) constructed wetland. Quantitative analysis of diclofenac concentrations in water solution and plant tissues was conducted by high performance liquid chromatography analysis after sample pre-treatment with solid-phase extraction and liquid extraction, respectively. The FWS with Cyperus alternifolius obtained a maximum 69.3% diclofenac removal efficiency, while a control system without plant only had a removal efficiency of 2.7% at the end of the experiment period of 70 days. Based on mass balance study of the experimental system, it was estimated that plant uptake and in-plant conversion of diclofenac contributed about 21.4% of the total diclofenac removal in the mesocosm while the remaining 78.6% diclofenac was eliminated through biotic and abiotic conversion of diclofenac in the water phase. Diclofenac on the root surface and in roots, stems and leaves of Cyperus alternifolius was found at the concentrations of 0.15-2.59 μg/g, 0.21-2.66 μg/g, 0.06-0.53 μg/g, and 0.005-0.02 μg/g of fresh weight of plant tissues, respectively. The maximum bioaccumulation factor of diclofenac was calculated in roots (21.04) followed by root surface (20.49), stems (4.19), and leaves (0.16), respectively. Diclofenac translocation potentiality from root to stem was found below 0.5, suggesting a slow and passive translocation process of diclofenac. Current study demonstrated high potential of Cyperus alternifolius for phytoremediation of diclofenac in FWS and can be applied in other engineered ecosystems.

Vibrio cholerae, the etiological agent of cholera, has been a scourge for centuries. Cholera remains a serious health threat for developing countries and has been responsible for millions of deaths globally over the past 200 years. Identification of V. cholerae has been accomplished using a variety of methods, ranging from phenotypic strategies to DNA based molecular typing and currently whole genomic approaches. This array of methods has been adopted in epidemiological investigations, either singly or in the aggregate, and more recently for evolutionary analyses of V. cholerae. Because the new technologies have been developed at an ever increasing pace, this review of the range of fingerprinting strategies, their relative advantages and limitations, and cholera case studies was undertaken. The task was challenging, considering the vast amount of the information available. To assist the study, key references representative of several areas of research are provided with the intent to provide readers with a comprehensive view of recent advances in the molecular epidemiology of V. cholerae. Suggestions for ways to obviate many of the current limitations of typing techniques are also provided. In summary, a comparative report has been prepared that includes the range from traditional typing to whole genomic strategies.

A poly(vinyledene difluoride)-lithium bis(oxalato)borate solid polymer electrolyte prepared by a solvent casting method has been irradiated with different doses of gamma-rays. Differential scanning calorimetry reveals that the polymer electrolyte irradiated with 35 kGy of γ-rays is the most amorphous sample. This is also supported by the results obtained from X-ray diffraction. The Fourier transform infrared spectrum of each irradiated sample has been deconvoluted in the wavenumber region between 1830 and 1758 cm(-1) in order to predict the percentage of free and contact ions in the samples. The sample exposed to 35 kGy of γ-rays contains the highest percentage of free ions and the lowest amount of contact ions. This sample also exhibits the highest room temperature conductivity of 3.05 × 10(-4) S cm(-1), which is 15% higher relative to the virgin sample. The number density of free ions is observed to have more control on the conductivity variation with the γ-radiation dose compared to ionic mobility. This study confirms that γ-irradiation can be a potential way to obtain highly conductive and mechanically stable polymer electrolytes.

Interactomic data for Kaposi's Sarcoma Associated Herpes virus (KSHV)-the causative agent of vascular origin tumor called Kaposi's sarcoma-is relatively modest to date. The objective of this study was to assign functions to the previously uncharacterized ORFs in the virus using computational approaches and subsequently fit them to the host interactome landscape on protein, gene, and cellular level. On the basis of expression data, predicted RNA interference data, reported experimental data, and sequence based functional annotation we also tried to hypothesize the ORFs role in lytic and latent cycle during viral infection. We studied 17 previously uncharacterized ORFs in KSHV and the host-virus interplay seems to work in three major functional pathways-cell division, transport, metabolic and enzymatic in general. Studying the host-virus crosstalk for lytic phase predicts ORF 10 and ORF 11 as a predicted virus hub whereas PCNA is predicted as a host hub. On the other hand, ORF31 has been predicted as a latent phase inducible protein. KSHV invests a lion's share of its coding potential to suppress host immune response; various inflammatory mediators such as IFN-γ, TNF, IL-6, and IL-8 are negatively regulated by the ORFs while Il-10 secretion is stimulated in contrast. Although, like any other computational prediction, the study requires further validation, keeping into account the reproducibility and vast sample size of the systems biology approach the study allows us to propose an integrated network for host-virus interaction with good confidence. We hope that the study, in the long run, would help us identify effective dug against potential molecular targets.

It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol(-1), whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol(-1). A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.

Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8-naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 degrees C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25 degrees C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 degrees C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.