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Huperzine A is an alkaloid featuring a bicyclo [3.3.1] nonane scaffold with an integrated piperidine ring, which was firstly isolated from Huperzia selago in 1960. As a reversible acetylcholinesterase inhibitor, it was clinically approved in China for treatment of Alzheimer's disease in 1996. Although huperzine A shows therapeutic potential, it is often associated with adverse events, such as dizziness, nausea, and digestive disorders. To enhance the efficacy and therapeutic index of huperzine A, many structural modification efforts have been undertaken. This review comprehensively summarizes the structural modification investigations on huperzine A conducted over the past three decades, and the structure-activity relationships are also discussed. The insights from this review are expected to inspire more effective modification strategies in the future., 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 Inc. All rights reserved.)

Oncogenes and tumor suppressors are well-known to orchestrate several signaling cascades, regulate extracellular and intracellular stimuli, and ultimately control the fate of cancer cells. Accumulating evidence has recently revealed that perturbation of these key modulators by mutations or abnormal protein expressions are closely associated with drug resistance in cancer therapy; however, the inherent drug resistance or compensatory mechanism remains to be clarified for targeted drug discovery. Thus, dual-target drug development has been widely reported to be a promising therapeutic strategy for improving drug efficiency or overcoming resistance mechanisms. In this review, we provide an overview of the therapeutic strategies of dual-target drugs, especially focusing on pharmacological small-molecule compounds in cancer, including small molecules targeting mutation resistance, compensatory mechanisms, synthetic lethality, synergistic effects, and other new emerging strategies. Together, these therapeutic strategies of dual-target drugs would shed light on discovering more novel candidate small-molecule drugs for the future cancer treatment., (© 2024 Wiley Periodicals LLC.)

A series of C-3 arylated huperzine A (HPA) derivatives (1-30) were designed and synthesized in good yields via palladium-catalyzed Suzuki cross-coupling reaction. Cholinesterase inhibitory and neuroprotective activities of all 30 derivatives were evaluated. Cholinesterase inhibition results revealed that derivatives 2 and 15 exhibited dual inhibitory activity against both acetylcholinesterase (AChE inhibition: 2, IC 50  = 1.205 ± 0.395 μM; 15, IC 50  = 0.225 ± 0.062 μM) and butyrylcholinesterase (BChE inhibition: 2, IC 50  = 8.598 ± 3.605 μM; 15, IC 50  = 4.013 ± 0.068 μM), a feature not observed in huperzine A. Molecular docking results indicated that the introduction of aryl groups enhanced the affinity of the derivatives for the acyl-binding pocket of BChE, thereby limiting the hydrolysis of acetyl choline. However, these derivatives exhibited poor performance in cytotoxicity and neuroprotection assays., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024. Published by Elsevier B.V.)

Bioinspired skeleton transformation of a tricyclic lathyrane-type Euphorbia diterpene was conducted to efficiently construct a tetracyclic tigliane diterpene on a gram scale via a key aldol condensation. The tigliane diterpene was then respectively converted into naturally rare ingenane and rhamnofolane diterpenes through a semipinacol rearrangement and a visible-light-promoted regioselective cyclopropane ring-opening reaction. This work provides a concise strategy for high-efficiency access to diverse polycyclic Euphorbia diterpene skeletons from abundant lathyrane-type natural products and paves the way for biological activity investigation of naturally rare molecules.

Aim: To design and synthesize a novel series of 1-aryldonepezil analogues. Materials & methods: The 1-aryldonepezil analogues were synthesized through palladium/PCy 3 -catalyzed Suzuki reaction and were evaluated for cholinesterase inhibitory activities and neuroprotective effects. In silico docking of the most effective compound was conducted. Results: The 4- tert -butylphenyl analogue exhibited good inhibitory potency against acetylcholinesterase and butyrylcholinesterase and had a favorable neuroprotective effect on H 2 O 2 -induced SH-SY5Y cell injury. Conclusion: The 4- tert -butylphenyl derivative is a promising lead compound for anti-Alzheimer's disease drug development.

A series of novel N -aryl-debenzeyldonepezil derivatives ( 1-26 ) were designed and synthesized as cholinesterase inhibitors by the modification of anti-Alzheimer's disease drug donepezil, using Palladium catalyzed Buchwald-Hartwig cross-coupling reaction as a key chemical synthesis strategy. In vitro cholinesterase inhibition studies demonstrated that the majority of synthesized compounds exhibited high selective inhibition of AChE. Among them, analogue 13 possessing a quinoline functional group showed the most potent AChE inhibition effect and significant neuroprotective effect against H 2 O 2 -induced injury in SH-SY5Y cells. Furthermore, Compound 13 did not show significant cytotoxicity on SH-SY5Y. These results suggest that 13 is a potential multifunctional active molecule for treating Alzheimer's disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xu, Luo, Xi, Xu and Wan.)

Autophagy is well-known to be a lysosome-mediated catabolic process for maintaining cellular and organismal homeostasis, which has been established with many links to a variety of human diseases. Compared with the therapeutic strategy for inhibiting autophagy, activating autophagy seems to be another promising therapeutic strategy in several contexts. Hitherto, mounting efforts have been made to discover potent and selective small-molecule activators of autophagy to potentially treat human diseases. Thus, in this perspective, we focus on summarizing the complicated relationships between defective autophagy and human diseases, and further discuss the updated progress of a series of small-molecule activators targeting autophagy in human diseases. Taken together, these inspiring findings would provide a clue on discovering more small-molecule activators of autophagy as targeted candidate drugs for potential therapeutic purposes., 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 Masson SAS. All rights reserved.)

A series of arylated huperzine A (HPA) derivatives ( 1 - 24 ) were efficiently synthesized in good yields (45-88% yields) through the late-stage modification of structurally complex natural anti-Alzheimer's disease (AD) drug huperzine A (HPA), using the palladium-catalyzed Suzuki-Miyaura cross-coupling reaction. The acetylcholinesterase (AChE) inhibitory activity of all synthesized compounds was evaluated to screen the potential anti-AD bioactive molecules. The results showed that introducing the aryl groups to C-1 position of HPA resulted in the unsatisfactory AChE inhibitory activity. The present study demonstrably verifies pyridone carbonyl group could be the necessary and unchangeable pharmacophore for maintaining HPA's anti-AChE potency, and provides the helpful information on the further research for developing anti-AD HPA analogues.

A series of new N -aryl galantamine analogues ( 5a - 5x ) were designed and synthesized by modification of galantamine, using Pd-catalyzed Buchwald-Hartwig cross-coupling reaction in good to excellent yields. The cholinesterase inhibitory and neuroprotective activities of N -aryl derivatives of galantamine were evaluated. Among the synthesized compounds, the 4-methoxylpyridine-galantamine derivative ( 5q ) (IC 50 = 0.19 μM) exhibited excellent acetylcholinesterase inhibition activity, as well as significant neuroprotective effect against H 2 O 2 -induced injury in SH-SY5Y cells. Molecular docking, staining, and Western blotting analyses were performed to demonstrate the mechanism of action of 5q . Derivative 5q would be a promising multifunctional lead compound for the treatment of Alzheimer's disease.

The organocatalytic asymmetric Morita-Baylis-Hillman (MBH) reaction of isatin derivatives with various vinyl sulfones is disclosed. Chiral sulfone-containing 3-hydroxyoxindoles were produced in good to high yields and with good to high ee's. This report displays an unprecedented example to apply activated alkenes with sulfone moiety other than carbonyl groups in asymmetric MBH reactions and provides an efficient strategy to incorporate the sulfone functional group for the synthesis of chiral 3-hydroxyoxindoles.

The biorelevant sulfur-containing Euphorbia diterpenes with scarce 5/7/6/3 premyrsinane- and 5/7/6 myrsinane-type backbones were easily constructed from naturally abundant lathyrane-type Euphorbia factor L 3 by visible-light-triggered tandem thiol-ene click reaction/transannular cyclization and regioselective cyclopropane ring-opening. The selenide diterpene was also successfully obtained to verify the system universality. This concise synthesis route gives an efficient strategy for obtaining structurally diverse Euphorbia diterpenes under very mild conditions and provides a promising anti-HIV bioactive premyrsinane diterpene 3h .

This brief proposes a signal-independent background calibration in pipeline-SAR analog-to-digital converters (ADCs) with a convergence-accelerated technique. To achieve signal independence, an auxiliary capacitor array C A is introduced to pre-inject a pseudo-random noise (PN) in the sampling phase to cancel out the opposite PN injection of the calibrated capacitor in the conversion phase, and C A is also used to realize the D/A function of the calibrated capacitor in the conversion phase. In this way, no matter what the signal is, the residue headroom remains unchanged even with PN injection. Moreover, the first sub-ADC is designed with extended conversion bits to quantize its own residue after delivering the conversion bits required by the first stage. Afterwards, this result is provided to the calibration algorithm to reduce the signal component and accelerate the convergence. Based on the simulation, the signal-to-noise and distortion ratio (SNDR) and spur-free dynamic range (SFDR) improve from 45.3 dB and 56.4 dB to 68.2 dB and 88.4 dB, respectively, after calibration. In addition, with the acceleration technique, convergence cycles decrease from 1.7 × 10 8 to 5.8 × 10 6 . Moreover, no matter whether the input signal is DC, sine wave or band-limited white noise, the calibration all works normally.

Broussonetia papyrifera , an important fast-growing economic tree species in China, has the advantages of strong adaptability, high-biomass, and high bioconcentration of heavy metals. Sewage sludge contains a great deal of nutrients and heavy metals. Planting B. papyrifera with sewage sludge can achieve the goals of sewage sludge remediation as well as resources production of B. papyrifera . A pot experiment was conducted to investigate growth, uptake and accumulation of nutrient and heavy metal in different organs (root, stem, leaf) of B. papyrifera , with treatments of control (lateritic red soil), 50% sewage sludge (mixed substrates of 50% sewage sludge and 50% lateritic red soil based on weight) and 100% sewage sludge. The comprehensive evaluation of capacity of uptake and accumulation was also carried out by principal component analysis and membership function. The results showed that B. papyrifera could grow normally in both 50% and 100% sewage sludge substrates, with higher plant height and biomass than that in the control, especially in 100% sewage sludge substrate. The quality index in 100% sewage sludge substrate (1.02) was 4.3 times and 2.4 times as that of the control and 50% sewage sludge substrate, respectively. The content of N in different organs and P in stem increased significantly in both 50% and 100% sewage sludge substrates. The content of K in stem and leaf was significantly decreased in 100% sewage sludge substrate, which were significant lower than that of control. The uptake of heavy metals such as Cu, Zn, Pb, Cd, Ni for B. papyrifera were mainly through roots. There was positive correlation between the content of heavy metals in root and sewage sludge ratio. The content of Pb and Cd in leaves were lower than the limit value of Hygienic Standard For Feeds (GB 13078-2017). The capacity for absorption and accumulation of Cd was better than that of other heavy metals. Compared with the control, rootretention rates of Zn, Pb and Cd significantly increased in both 50% and 100% sewage sludge substrates (57.8%-85.8%), while Cu and Ni significantly increased in 100% sewage sludge substrate (67.5% and 74.8%). Nutrient and heavy metal accumulations in total plant in both 50% and 100% sewage sludge substrates were significantly higher than that in the control, with 100% sewage sludge substrate being significantly higher than that in 50% sewage sludge substrate. Compared with 50% sewage sludge substrate, the increment rates of nutrient and heavy metal accumulations in different organs as well as total plants in 100% sewage sludge substrates were greatly increased. The rank of comprehensive evaluation scores of adaptability, element uptake and accumulation was in an order: 100% sewage sludge substrate (0.848) > 50% sewage sludge substrate (0.344) > control (0.080). With good adaptability to sewage sludge, B. papyrifera could grow normally in sewage sludge andeffectively absorb and fix nutrients and heavy metals. It is feasible to plant B. papyrifera into the sewage sludge for remediation of sewage sludge and resource production.

Large-scale, highly integrated and low-power-consuming hardware is becoming progressively more important for realizing optical neural networks (ONNs) capable of advanced optical computing. Traditional experimental implementations need N 2 units such as Mach-Zehnder interferometers (MZIs) for an input dimension N to realize typical computing operations (convolutions and matrix multiplication), resulting in limited scalability and consuming excessive power. Here, we propose the integrated diffractive optical network for implementing parallel Fourier transforms, convolution operations and application-specific optical computing using two ultracompact diffractive cells (Fourier transform operation) and only N MZIs. The footprint and energy consumption scales linearly with the input data dimension, instead of the quadratic scaling in the traditional ONN framework. A ~10-fold reduction in both footprint and energy consumption, as well as equal high accuracy with previous MZI-based ONNs was experimentally achieved for computations performed on the MNIST and Fashion-MNIST datasets. The integrated diffractive optical network (IDNN) chip demonstrates a promising avenue towards scalable and low-power-consumption optical computational chips for optical-artificial-intelligence., (© 2022. The Author(s).)

Palladium/BuAd 2 P efficiently catalyzed the direct α-arylation of ketone in the anti-Alzheimer's disease drug donepezil, leading to 15 aryldonepezil analogues exhibiting high selective inhibition of acetylcholinesterase (AChE). The cell-based assays revealed that the 3-methylpridinyl analogue ( 12 ) shows significantly lower toxicity compared to donepezil and remarkable neuroprotective activity against H 2 O 2 -induced damage in SH-SY5Y cells. Docking results of compound 12 also interpreted the possible mechanism of the selective inhibition between AChE and butyrylcholinesterase (BuChE)., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The first systematic direct diversification of a complex natural product by metal-catalyzed N-H functionalization was carried out. A new series of N -(hetero)aryl analogues ( 1 - 32 ) of the natural anti-Alzheimer's disease drug huperzine A (HPA) was prepared via palladium-catalyzed Buchwald-Hartwig cross-coupling reactions of HPA with various aryl bromides in good yields. Most of the N -aryl-huperzine A ( N -aryl-HPA) analogues showed good acetylcholinesterase (AChE) inhibitory activity in in vitro experiments. Three arylated huperzine A analogues ( 14 , 19 , and 30 ) exhibited stronger anti-AChE activity than HPA. The 5-methoxy-2-pyridyl analogue ( 30 ) displayed the most potent AChE inhibition activity, with an IC 50 value of 1.5 μM, which was 7.6-fold more active than HPA. Compound 30 also exhibited better neuroprotective activity for H 2 O 2 -induced damage in SH-SY5Y cells than HPA. Structure-activity relationship analysis suggested that the electron density of the installed aromatic ring or heteroaromatic ring played a significant role in inducing the AChE inhibition activity. Overall, compound 30 showed the advantages of easy synthesis, high potency and selectivity, and improved neuroprotection, making it a potential huperzine-type lead compound for Alzheimer's disease drug development.

Two new premyrsinane-type diterpenes ( 2 and 3 ) as diastereomers were synthesized from lathyrane-type diterpene euphorbia factor L 3 ( 1 ) for the first time via an efficient Fe(acac) 3 -catalyzed skeleton conversion process. This conversion features a biogenetically inspired strategy that relies on a concise reductive olefin coupling involving intramolecular Michael addition with free radicals. The structures of 2 and 3 were elucidated by a combination of the interpretation of their spectroscopic data and single-crystal X-ray diffraction analysis. The premyrsinane diterpenes 2 and 3 exhibited cytotoxic activity against the 4T1 breast cancer cell line, while the parent compound euphorbia factor L 3 ( 1 ) was inactive. The current results not only confirmed the biogenetic relationship between lathyranes and premyrsinanes for the first time but also suggested a novel method for the preparation of naturally rare premyrsinane diterpenes with high bioactivity from the more abundant natural lathyrane diterpenes.

Five new aconitine-type C 19 -diterpenoid alkaloids, apetalrines A-E ( 1 - 5 ), were isolated from Aconitum apetalum . Their structures were determined by analysis of 1D and 2D NMR, IR, and HRESIMS data. Semisynthesis of apetalrine B ( 2 ) from its parent compound aconorine was achieved to confirm the structure proposed. Twenty derivatives of 2 ( 11a - 11l , 12a , 12b , 12d , 12e , 12j , 12k , 12m , 12n ) were synthesized via a unified approach relying on simple coupling reactions. The evaluation of neuroprotective effects of compounds ( 1 - 5 , 11b , 11c , 11f - 11i , 12a , 12b , 12d , 12e , 12k , 12m , 12n ) with low cytotoxicity revealed compound 2 to exhibit good neuroprotective effects in H 2 O 2 -treated SH-SY5Y cells at a concentration of 50 μM. A series of studies using flow cytometry, staining, and Western blotting on 2 indicated that its neuroprotective effects may arise from inhibiting cell apoptosis.

A new series of N -aryltacrine derivatives were designed and synthesized as cholinesterase inhibitors by the late-stage modification of tacrine, using the palladium-catalyzed Buchwald-Hartwig cross-coupling reaction. In vitro inhibition assay against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) demonstrated that most of the synthesized compounds had potent AChE inhibitory activity with negative inhibition of BuChE. Among them, N -(4-(trifluoromethyl)phenyl)-tacrine ( 3g ) and N -(4-methoxypyridin-2-yl)-tacrine ( 3o ) showed the most potent activity against AChE (IC 50 values of 1.77 and 1.48 μM, respectively). The anti-AChE activity of 3g and 3o was 3.5 times more than that of tacrine (IC 50 value of 5.16 μM). Compound 3o also displayed anti-BuChE activity with an IC 50 value of 19.00 μM. Cell-based assays against HepG2 and SH-SY5Y cell lines revealed that 3o had significantly lower hepatotoxicity compared to tacrine, with additional neuroprotective activity against H 2 O 2 -induced damage in SH-SY5Y cells. The advantages including synthetic accessibility, high potency, low toxicity, and adjunctive neuroprotective activity make compound 3o a new promising multifunctional candidate for the treatment of Alzheimer's disease., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Objective: To investigate the effect and mechanism of hyperbaric oxygen combined with radioactive seed implantation in the treatment of esophageal squamous cell carcinoma. Methods: Subcutaneous tumor model of esophageal squamous cell carcinoma using TE-8 cells was established. Tumor bearing Balb/c(nu/nu) mice (60 mice) were divided into four groups, Cont group that treated with normal oxygen level, HBO group that treated with hyperbaric oxygen, RSI group that treated with radioactive seed implantation, and HBO+ RSI group that treated with hyperbaric oxygen combined with radioactive seed implantation. Tumor volume ratio and mean survival time of tumor bearing mice were observed. Pathological changes of tumor tissue after treatment were observed by hematoxylin eosin (HE) staining. Enzyme linked immunosorbent assay kit was used to detect oxidative stress. Apoptosis related proteins were detected by Western blot. Results: After treatment, the tumor volume ratio of HBO+ RSI group was 3.51±0.80 and was significantly lower than that of Cont group, HBO group, and RSI group ( P <0.05). The mean survival time of HBO+ RSI group tumor bearing mice was 62 d and was significantly longer than that in Cont group, HBO group, and RSI group ( P <0.05). HE staining showed that the pathological changes of tumor tissues were most obvious in HBO+ RSI group. After treatment, the MDA and Bax levels in nude mice of HBO+ RSI group were significantly higher than those in Cont group, HBO group and RSI group, but the levels of GSH, SOD and Bcl-2 were significantly lower than those of Cont group, HBO group and RSI group ( P <0.05). Conclusion: Hyperbaric oxygen combined with radioactive seed implantation could slow tumor growth and increase survival time of tumor bearing mice. The possible mechanism is that hyperbaric oxygen combined with radioactive seed implantation can improve the oxidative stress response and the expression of apoptosis protein in tumor bearing nude mice.

Objective: To evaluate the effects of comprehensive schistosomiasis control in marshlands of Pukou District, Nanjing City so as to provide the evidence for developing the strategy of the disease control., Methods: The information of the prevention and control of schistosomiasis was collected in Pukou District from 2004 to 2013. The variations of the infection rates of human, livestock and Oncomelania hupensis snails were analyzed., Results: In 2013, the area with snails in the entire district was 384.09 hm2, which was 66.24% decrease compared to 1 137.61 hm2 in 2004. Moreover, after 2008, no infected-snails and schistosomiasis patients were found, and both the density of live snails and serum positive rate of schistosomiasis declined with years., Conclusions: After the comprehensive prevention and control is conducted, the schistosomiasis situation in Pukou District is stable. However, in order to consolidate the results, it is still essential to continue monitoring the previous infected snail environments and implementing the comprehensive prevention and control measures.

Olfactory receptors are G protein-coupled receptors that mediate olfactory chemosensation and serve as chemosensors in other tissues. We find that Olfr78, an olfactory receptor expressed in the kidney, responds to short chain fatty acids (SCFAs). Olfr78 is expressed in the renal juxtaglomerular apparatus, where it mediates renin secretion in response to SCFAs. In addition, both Olfr78 and G protein-coupled receptor 41 (Gpr41), another SCFA receptor, are expressed in smooth muscle cells of small resistance vessels. Propionate, a SCFA shown to induce vasodilation ex vivo, produces an acute hypotensive response in wild-type mice. This effect is differentially modulated by disruption of Olfr78 and Gpr41 expression. SCFAs are end products of fermentation by the gut microbiota and are absorbed into the circulation. Antibiotic treatment reduces the biomass of the gut microbiota and elevates blood pressure in Olfr78 knockout mice. We conclude that SCFAs produced by the gut microbiota modulate blood pressure via Olfr78 and Gpr41.

Objective: To understand the status of Clonorchis sinensis infection and its risk factors among migrant workers in Baoan District of Shenzhen City, so as to provide the evidence for the development of control strategies., Methods: A total of 642 migrant workers were chosen as the investigation samples by the stratified cluster sampling method. Their sera were collected and tested for Clonorchis sinensis infection with ELISA, and a questionnaires survey was performed to collect the information of clonorchiasis sinensis related to knowledge and behaviors. The influencing factors were summarized with the case-control study method., Results: A total of 642 subjects were investigated, in which 530 subjects received the serological examinations and the positive rate was 6.6% (35/530). The significant differences were not found between genders (Chi2 = 1.19, P = 0.28) or among the age groups (Chi2 = 0.45, P = 0.80). The awareness rates of knowing infection route, health hazard and prevention knowledge were 50.16%, 33.64% and 27.41%, respectively. The rates of healthy behaviors such as not eating semi-cooked fish, not feeding pets with raw fish or shrimps, and differentiating between the raw and cooked food when using cutting boards were 80.67%, 78.41% and 45.95%, respectively. The awareness rate of prevention knowledge was positively related to the infection (OR = 0.16, P < 0.01)., Conclusion: The prevention and control of Clonorchis sinensis infection among migrant workers could not be neglected, and the health education should be strengthened.

The present study aimed to observe the therapeutic effect of high-intensity focused ultrasound for the treatment of allergic rhinitis (AR) using nasal endoscopy. A total of 72 patients with perennial AR received treatment using the CZB ultrasonic therapeutic instrument with nasal endoscopy. A scoring method was adopted for evaluation of effectiveness according to the AR therapeutic principles and recommendations described in Allergic Rhinitis and its Impact on Asthma (ARIA) in 2001. The patients were followed up between 2 and 6 months after treatment. The excellence rate was 34.7% (25/72), the effective rate was 62.5% (45/72) and the ineffective rate was 2.8% (2/72). The total effective rate reached 97.2% high (70/72). Endoscopic high-intensity focused ultrasound for the treatment of AR is a non-invasive method and has the advantages of simple manipulation, a short course, high safety and a clear short-term effect.

Objective: To master the epidemic situation and control effect of schistosomiasis in Pukou District, Nanjing City., Methods: The data of the epidemic situation and control measures of schistosomiasis in Pukou District from 2004 to 2011 were collected and analyzed for the trends of schistosomiasis epidemic situation and changes of status of Oncomelania snails., Results: In 2011, the area with snails and the area with infected snails were 383.11 hm2 and 0 hm2 respectively, the number of blocks with snails were 15, and all dropped substantially compared with those in 2004. The population serological positive rate was decreasing from 2004-2011., Conclusion: The schistosomiasis in Pukou District presents a low prevalence and the control measures are effective.