We engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krüppel. In addition, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a noncanonical role for Bicoid in directly suppressing knirps transcription. Acute modulation of transcription factor concentration while recording output gene activity represents a powerful approach for studying developmental gene networks in vivo.Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal the kinetics of gene expression downstream of a developmental transcription factor   kni repression.Singh et al. investigate how rapid changes in nuclear Bicoid levels dynamically regulate expression of its target genes. Combining optogenetic stimulation and a simplified genetic background, their results recapitulate the canonical role of Bicoid as a transcriptional activator and reveal a possible noncanonical role in direct  Gene networks play a crucial role in developmental patterning, transforming rudimentary positional cues into a multitude of sharply defined domains of gene expression. Such networks are typically characterized by redundant inputs, to ensure that gene expression is initialized appropriately, and feedback connections between genes in the network, to ensure a consistent patterning response. Information from these inputs is integrated at enhancers that bind multiple transcription factors and may control gene expression through transient interactions with promoters or longer-term alterations in chromatin structure and accessibility. Understanding how networks function requires knowing the time scales over which individual components operate.Drosophila embryo is a canonical example of such a sophisticated pattern-forming system. In this network, the expression of four core transcription factors—the gap genes giant (gt), hunchback (hb), Krüppel (Kr), and knirps (kni)—is initiated by three partially redundant sources of positional information that are maternally deposited in the egg. These maternally supplied inputs include an anterior-to-posterior gradient of the Bicoid (Bcd) transcription factor, a posterior-to-anterior gradient of the Nanos RNA binding protein, and Torso receptor tyrosine kinase signaling at the anterior and posterior poles. In addition to responding to maternal inputs, the four gap genes further regulate themselves and one another to generate bands of gene expression that are essential for specifying the body plan  in embryos harboring each of the three Bcd-LEXY constructs  and 2E. Drosophila embryogenesis. During preparation of this article, a LEXY variant harboring point mutations that slow down reversion to the dark state after photoactivation (Drosophila embryos (To assess nuclear-cytosolic translocation efficiency of our fluorescent protein (FP)-Bcd-LEXY constructs, we quantitatively characterized nuclear import and export dynamics for each construct as well as two LEXY-tagged fluorescent proteins that lacked any transcription factor fusions (NLS-mCherry-LEXY and NLS-EGFP-LEXY) . Comparativation  was repo embryos . Our app embryos . While bbcdE1nosBNtsl4/bcdE1nosl7tsl4 (henceforth referred to as bnt) embryos as a starting point for introducing light-controlled Bcd-LEXY constructs (see nos TCEIIUC:AG) .Our goal is to use the fly embryo as a laboratory to measure stimulus-response functions for Bcd’s regulation of gap gene expression. We thus sought to simplify the experimental system, eliminating redundant inputs to the gap gene network as well as the complex spatial patterns found in wild-type embryos. Recent studies  establisIIUC:AG)  to supprIIUC:AG) ; the resh 3′ UTR . Bcd-LEXgt, hb, Kr, and kni)  drive expression of 24xMS2 stem loops followed by the yellow gene .To define the A-P position represented by each uniformly expressed Bcd-LEXY construct, we measured the transcriptional activity for four gap genes (kni) see . We usedlow gene . We conflow gene  and S2C.nos-tub bnt embryos and how it was perturbed by the addition of different optogenetic Bcd constructs. nos-tub bnt embryos transcribed high levels of kni MS2 and gt MS2, but low levels of Kr MS2 and no detectable hb MS2  upon illumination. Similarly, iRFP-Bcd-LEXY embryos shift from ~45% to 55% EL upon illumination, and mCherry-Bcd-LEXY embryos from ~50% to 60% EL , hb (iRFP-Bcd-LEXY), Kr (Bcd-LEXY), and kni (mCherry-Bcd-LEXY)  laser for GFP/mCherry imaging with a digital micromirror device and 450 nm LED for optogenetic stimulation see  and S3A.gt MS2 reporter). When appropriate combinations of Bcd-LEXY constructs were co-expressed with gap gene MS2 reporters, we found that local illumination was sufficient to drive sharp boundaries of localized gene expression at any user-defined embryonic position  expression  and repressed by Hb  still showed potent regulation by mCherry-Bcd-LEXY, whereas the kni Δdistal reporter was not affected by light-induced changes in nuclear Bcd  and four output genes , it captures much of the complexity of the wild-type pattern, including stripes of gap gene expression when Bcd is delivered in a head-to-tail gradient  or weak activation by Cad, which is translationally repressed by Bcd -target gene pairs in complex regulatory networks.How might rapid transcriptional activation occur upon loss of nuclear Bcd? Our data are consistent with many possible mechanisms. Bcd may compete for binding to the ral axis . We lookin vivo. Nevertheless, work on an experimentally reduced system constitutes only a first step in understanding the full gap gene network, and we look forward to future studies that examine Bcd-dependent responses as other factors from the natural system are systematically reintroduced. We can also envision extending the current approach to perturbing multiple nodes , and coupling these approaches to quantitative modeling . A second limitation is that we primarily consider Bcd’s role as a TF, not its additional role as a translational repressor of Cad protein. Alternative optogenetic strategies and live-cell Cad biosensors  could altoettcher@princeton.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jared Toettcher  digested by restriction enzymes NheI and SpeI. mCherry was subsequently replaced by EGFP to generate NLSEGFP-LEXY plasmid.Constructs were generated using In-Fusion assembly (Clontech) and oligonucleotides for primers were obtained from Integrated DNA Technologies. Constructs are available via Addgene or on request. Bcd-LEXY constructs are generated from pCol-aqh 3’UTR  where thqh 3’UTR  and a flbcdE1 or bcdE1nosBNtsl4 on the third chromosome  constructs were integrated into the third chromosome using the φC31-based integration system  at the Vromosome  NLS-mChebcdE1nosBNtsl4 homozygous females, and thus used bcdE1nosBNtsl4/bcdE1nosl7tsl4 transheterozygotes for further experiments. While nosBN is a complete loss of both nos RNA and protein, which impedes both pole cell migration and therefore germline cells formation and abdominal segmentation, nosl7 is a partial deletion near C-terminal of the zinc-finger domain that maintains normal germline development. However, in terms of body segmentation phenotype and gap gene expression pattern, nosBN and nosl7 both exhibit indistinguishable patterns as expected from severe loss of function  to mark the transgene , and then crossed with male uniform Bcd-LEXY bcdE1, ri, nosBN,e, tsl4/TM3, sb, ri flies to generate Sp, nos-tub/+; uBcd-LEXY bcdE1, ri, nosBN, e, tsl4 / TM3, sb, ri flies. By crossing males of the preceding genotype to MCP-mNeonGreen, NLS-mCherry-LEXY / Cyo; bcdE1, ri, nosl7tsl4 / TM3,sb, ri females, we selected female flies with Sp and ri markers to ensure the correct genotype of Sp, nos-tub / MCP-mNeonGreen NLS-mCherry-LEXY; uBcd-LEXY bcdE1, ri, nosBN, e, tsl4 / bcdE1, ri, nosl7tsl4 that we then caged with homozygous MS2 reporter males for live gap gene transcription imaging.In wild-type embryos,  protein  A secondnslation  Completeonstruct  (courteshb BAC>MS2 (BAC CH322–55J23) and kni BAC>MS2 (BAC CH322–21P08) were described in Bothma et al., where CHORI BACs (~21kb) were used as starting points, with kni and hb coding sequences replaced with a 24xMS2-yellow-kanamycin reporter gene, leaving the 5’UTR and 3’UTR intact. kni Δdistal and Δproximal MS2 reporters were also described previously >MS2 was described in El-Sherif et al., where a 4kb upstream regulatory region including promoter region is fused to 24xMS2-yellow reporter  for another 24–36 h and then bleached, then moved to the methanol-heptane glass tube and vigorously shaken for 20 s. Embryos settled at the bottom were removed and placed on a glass slide with Hoyer’s solution (1:1 premix lactic acid) and sandwiched between the glass slide and cover glass. The slide was placed at 65°C overnight and then imaged on a Nikon Eclipse Ni dark-field microscope at 10x zoom.Embryos were collected every 2 h and aged in dark for another 2 h. Embryos were dechorionated by bleaching and heat fixed in dark, and then stained essentially as described in  with rabA custom microscope was built to simultaneously perform two-photon excitation imaging and localized optogenetic stimulation on the same setup. A Chameleon Ultra II tunable laser was used at 970 nm to simultaneously excite green and red-tagged biomolecules  The lase2 of blue-light on/off pulsatile cycles of LEXY-tagged protein nuclear signal (data not shown), 100 μW/cm2  was determined for all optogenetic perturbations performed in this study.LEXY perturbation was achieved using a digital micro-mirror device  to project spatial patterns and to rapidly change light levels  see Fig through For the live data acquisition and light perturbation experiments, flies were kept in dark at 25°C and the embryos were collected on an agar plate between 1 and 2 h post laying. For live imaging, embryos were dechorionated on double-sided tape and mounted on a glued membrane film , covered in halocarbon oil 27 and sandwiched between the membrane and the cover glass slide . The data collection was performed using a custom-built two-photon microscope using 970 nm laser excitation for green (EGFP and mNeonGreen tagged proteins) and red (NLS-mCherry-LEXY) at room temperature (ranging from 21.5–22.5°C). The blue light perturbation was performed using a digital micromirror device (DMD) unit installed on the same system (see bcdE1 homozygous mutant fly lines and the Bcd dose level), we used protein immunostaining of the Bcd target gene Hb and measured position shifts in the posterior Hb boundary as well as position shifts of the cephalic furrow. All shifts were scaled according to embryo length and the quantified estimates are presented in To estimate the functional Bicoid activity (potency) of fluorescently tagged Bcd-LEXY fusion proteins -induced import kinetics were determined by analyzing the nuclear intensity of the fluorescent moiety of these fusion proteins. Intensity time traces were averaged and fitted with a single exponential ng model –2H. Noteng model . For LEX2 ROI after thresholding in the center of the embryo; similar results were also obtained if the mean intensity of foci was calculated instead, albeit with greater noise when the number of detected foci was low  from the mean of all embryos in the corresponding control condition  (The response time workflow is shown in t light) . The rest light)  and difft light) . The “re1Video S1Video S2Video S3


Instron Labtronic 8800, the operator controlled the tests at a frequency of 4,000 Hz. The data acquisition rate was 25,000 Hz, and for faster experiments, 100,000 Hz. The operator controlled on a random basis the hydraulic cylinder by either the cylinder displacement or the specimen displacement. Increasing as well as constant and decreasing compression strength trends with increasing strain rates could be shown in the past. The data presented here show a lower compressive strength at strain rates higher than 4*10−3 s−1. The data consist of the time history of the specimen and cylinder displacement measurement (in mm), and the force measurement (in kN). The data is available as a separate .xlsx file for each test performed. In total, 123 tests were performed. If the test was performed with a 10 mm gap, the label ends with a ‘g’. The abbreviations are separated with an underscore.Uniaxial compressive tests of laboratory-made granular fresh-water ice were conducted in a cold room in the ductile and brittle strain rate range at -10.83°C ±0.74°C. Ice specimens with a length to diameter ratio of 2.5 showing brittle behavior failed by axial splitting. With the The data provided here can be used to validate ice-material models or for ice-testing databases for machine learning purposes. The strength values in the brittle range are far below the strength values of Shazly et al. Ice specimens with a length to diameter ratio of 2.5 showing brittle behavior failed by axial splitting. The data presented here show a lower compressive strength from strain rates higher than 4*102The experimental design included the PID controller settings and the displacement control type and consisted of determining the compressive strength at high rates. The rate limitation was the Schenck Hydropuls-longitudinal PL 160 cylinder with its force capability of nominal 160 kN and a maximal velocity of 1 m/s. The hydraulic cylinder can be controlled either by the piston displacement  or by the specimen displacement (SDC). The specimen displacement controlled tests neglect the effect of the test rig stiffness. All tests are performed at TUHH in the mechanical laboratory of the Institute for Ship Structural Design and Analysis.2.1Cylindrical ice specimens with a diameter of 99.4 mm and a total length of 250 mm were produced in an approximately -10°C cold refrigerated container.PVC-U tubes, commercially available crushed ice, distilled water, steel plates, and insulating material were needed to make granular ice specimens. The distilled water had a mean acidity of 8.6 pH ±1 pH and a mean electrical conductivity of 7.5 μS/cm ± 5.8 μS/cm measured at a random basis at temperatures of 18.8°C ± 3.6°C.The tubes were cut into the desired length, including a minimum addition of 30 mm regarding the cutting process after the specimen removal, and glued to the steel plate on one side. The steel plate formed the base and ensured good heat conduction. In the refrigerated container, two-thirds of crushed ice and one-third of distilled water was added to the tube, and the top of the tube was covered with insulation material. After freezing for at least two days, the ice specimen was removed from the tube by heating it with a hot air gun up  for a short time. After removal, the ice specimens were stored in the refrigerated container and were cut at both ends into the desired length with a band saw located in the refrigerated container. This process ensured consistency in the grain structure and shape of the specimens. Herrnring et al. 2.2Schenk Hydropuls-Longitudinal PL 160 cylinder, the position of the load cell, and the position of the potentiometric displacement sensor burster 8718-500 for the specimen displacement measurement are shown in The Instron Labtronic 8800, the operator controlled the tests at a frequency of 4,000 Hz. The measurements are carried out with the Gantner Q.raxx-station 101 T measuring amplifier. The displacement, the force and the temperature are measured with the Q.raxx A101 module. The data acquisition rate was 25,000 Hz for test series 1 – 3 and 100,000 Hz for tests series 4, see also With the Angelo Mario Böhm: Conceptualization, Methodology, Investigation, Data Curation, Writing – original draft, Writing – review & editing, Visualization; Hauke Herrnring: Software, Writing – review & editing; Franz von Bock und Polach: Supervision, Writing – review & editing.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.


Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the pers phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the  Drosophila, the circadian clock comprises four core components (period (per) and timeless (tim) genes to promote their transcription. PER and TIM heterodimerize in the cytoplasm before entering the nucleus. Once inside the nucleus, the PER/TIM heterodimer binds to CLK/CYC and thus inhibits per and tim transcription. PER, TIM, and CLK undergo various post-translational modifications that affect the period of the clock, the most notable being the progressive phosphorylation driven by kinases such as DOUBLETIME (DBT)  , SHAGGY ME (DBT) , ProlineME (DBT) , CASEIN ME (DBT) , CASEIN ME (DBT) , AMPK (CME (DBT)  and NEMOME (DBT) .Drosophila pseudoobscura  binding. CCA1 binding to its target promoters increases with temperature, which is kept in check by the opposing effect of CK2 phosphorylation  to Tyrosine (Y) substitution at the 45th residue, which is a part of Drosophila PER’s phosphodegron  (short (pers)per phosphocluster, which is equivalent to the mammalian FASPS domain  results in an overcompensated circadian period, i.e., circadian period lengthens when ambient temperature increases. This overcompensation phenotype correlates with increased hyper-phosphorylated PER levels in vivo at warm temperature and overcompensated PER degradation in a well-established cell culture model. These results converge to support an important role of the phosphodegron in temperature compensation in Drosophila.Over the past several decades, circadian mutants have fuelled our understanding of the molecular clockwork. Therefore, to gain better understanding into the mechanism of temperature compensation in ensation . We were and 47) . The PER and 47) : it is p and 47) , and itsS domain . PhosphoS domain . SLIMB iSLIHper mutation  or E (Glutamate)] and phosphoinhibitors [S to Alanine (A)] at PER S44, S45, and S47 residues using CRISPR-CAS9  substitution to mimic contribution of a bulky hydrophobic amino acid similar to Tyrosine (Y) to validate previous observations on SLIHper. We used S45S as a control, which contains only the silent substitutions introduced at the protospacer adjacent motifs (PAM site) during CRISPR mutagenesis. In our hands white1118 (w1118) and S45S control strains showed a decrease in period of ∼0.9 h , but the effect on temperature compensation was difficult to interpret due to poor rhythmicity, particularly at 18°C . Howeverper genomic rescue constructs in an amorphic per0 background . S45D and S45E did not show any substantial effect on either period length or temperature compensation ; Table 1ckground . HoweverThus, taken together our results show that the PER phosphodegron modulates temperature compensation, and shows functional temperature compensation heterogeneity within its residues.pers domain (T/S583-596). Indeed, this region also undergoes phosphorylation by DBT and NMO, and phosphorylation in the pers domain temporally precedes and inhibits phosphorylation at S47  and pers (S589N substitution) mutations have also been reported to be undercompensated  flies showed rhythmic PER abundance and phosphorylation regardless of the temperature, albeit with significantly higher abundance at 29°C , but rhythmic locomotor behavior is controlled by a small number of circadian pacemaker neurons, the small ventral lateral neurons (sLNvs), that contribute minimally to whole-head protein extracts  in the phosphodegron increases temperature compensation, while in mammals mutation of the homologous S478 residue has the opposite effect ; PBac[y(+mDint2) = vas-Cas9]VK00027 (BL51324), y[1] M(Act5C-Cas9.P.RFP-)ZH-2A w[1118] DNAlig4[169] (BL58492). Transgenic flies used in Fly stocks were maintained on standard cornmeal agar at 25°C under 12:12 h light: dark (LD). The following available strains were used FM7a balancer flies. F1 progeny was screened with PCR and sequencing for the presence of the desired modifications. All the resultant lines were backcrossed into the w1118 genetic background. For lines generated from BL58492 , we determined whether the Cas9 transgene located on the X chromosome had been removed. S45E, S44D, and S47D fly lines unexpectedly retained Cas9 even after multiple attempts to remove it. It seems however highly unlikely that Cas9 could somehow interfere with temperature compensation, particularly since in both S45E and S44D temperature compensation was unaffected  was identified using CRISPR fly design. The guide RNA sequence chosen was—5′AGCCACTGCTGCCGGAGGAGTGG. The guide RNAs were cloned into plasmid pCFD3 . Silent modifications (AGC-AGT) were introduced into the seed sequence to prevent re-cutting and for subsequent molecular screening.Drosophila Activity Monitoring) system  in 136-LL incubators . Behavior was analyzed and plotted using FaasX software . Rhythmicity was defined by following the criteria: power >20, width >1.5 h, using the c2 periodogram analysis. 5 days of DD data were used to determine period. Period and power of all individual flies under various conditions can be found in supplemental file 2–5 days old male flies were loaded into behavior tubes. Flies were entrained at the defined temperature for at least 3 days under 12:12 LD cycle and released into constant darkness (DD). Activity was recorded using the DAM ] supplemented with 10% Fetal Bovine Serum (FBS) . Plasmids expressing pAc-per(WT)-V5, pAc-per(S45A)-V5, pAc-per(S47A)-V5, and pMT-dbt-V5 were described previously . For mobility shift assay (dbt-V5 and 0.8 μg of pAc-per(X)-V5, where X is either WT, S45A or S47A. dbt expression were induced with 500 μM CuSO4 36 h after transfection and cells were moved into incubators with indicated temperature and cells were harvested at 0, 6, 12, 24 h after induction. Proteins were analyzed by Western Blotting. For cycloheximide (CHX) chase experiment (dbt-V5 and 0.8 μg pAc-per(X)-V5, where X is either WT or S47A. dbt expression were induced with 500 μM CuSO4 at 36 h after transfection. 6 h after DBT induction, CHX (Sigma) was added at a final concentration of 10 μg/ml. Cells were harvested and lysed with EB2  at the indicated times. Proteins were analyzed by Western Blotting.eviously . For allft assay , S2 cellperiment , S2 cellDrosophila S2 cells and adult fly heads, western blotting, and image analysis was performed as previously described  at 1:3000 for PER-V5 and DBT-V5, α-PER  at 1:2000 for PER, α-HSP70 at 1:10000 for HSP70. Secondary antibodies conjugated with HRP were added as follows: α-mouse IgG (Sigma) at 1:2000 for α-V5 detection, 1:10000 for α-HSP70 detection, α-guinea pig IgG (Sigma) at 1:1000 for α-PER detection. All quantifications are included in supplemental file


Vending machines for harm reduction (VMHR) are an innovative approach to deliver life-saving materials, information, and treatment for hard-to-reach populations, particularly for persons who inject drugs. The current study explores stakeholders’ perspectives on the feasibility and acceptability of VMHR in Philadelphia.From October 2021 to February 2022, we conducted 31 semi-structured interviews with potential end users, staff, and leadership at a local federally qualified health center, and community members. Trained coders extracted themes from interview transcripts across four key domains: materials and logistics, location, access, and community introduction.Interviewees from all stakeholder groups endorsed using VMHR to provide supplies for wound care, fentanyl test strips, naloxone, and materials to connect individuals to treatment and other services. Dispensing syringes and medications for opioid use disorder were commonly endorsed by health center staff but were more controversial among potential end users. Even within stakeholder groups, views varied with respect to where to locate the machines, but most agreed that the machine should be placed in the highest drug use areas. Across stakeholder groups, interviewees suggested several strategies to introduce and gain community acceptance of VMHR, including community education, one-on-one conversations with community members, and coupling the machine with safe disposal of syringes and information to link individuals to treatment.Stakeholders were generally receptive to VMHR. The current study findings are consistent with qualitative analyses from outside of the USA and contribute new ideas regarding the anticipated community response and best methods for introducing these machines to a community. With thoughtful planning and design, VMHR could be a feasible and acceptable modality to reduce death and disease transmission associated with the opioid and HIV epidemics in cities like Philadelphia.The online version contains supplementary material available at 10.1186/s12954-023-00747-4. The opioid overdose epidemic in the USA continues unabated, further exacerbated by the rise of synthetic opioids and the COVID-19 pandemic . Fatal oTo alleviate pandemic-related treatment disruptions, public health officials also called for an expanded investment in harm reduction, an approach that emphasizes pragmatic strategies to help individuals mitigate the risk associated with their substance use and increase treatment readiness , 23. TheA promising and innovative harm reduction intervention that has emerged in the last fifteen years is the vending machine for harm reduction (VMHR). VMHR is an umbrella term for a range of mechanized devices that deliver materials, information, and treatment to historically excluded populations, particularly for people who inject drugs (PWID) , 35. AltDespite their promise, VMHR can introduce many multifaceted and contradictory feasibility and acceptability concerns. Stakeholders in qualitative studies of VMHR consider a number of practical questions, such as where the machines would be placed, how to keep the machines stocked, what supplies the machines would dispense, and whether the items should be free or have a cost , 27. ForThe lack of acceptability in different communities may obstruct initial or sustained implementation of VMHR, which is concerning given that attitudes toward VHMR can vary widely across stakeholder groups , 28–30. In the USA and Canada, there are VMHR available for rental or purchase that are already in use in several jurisdictions. They range from providing only one item  to providing a number of items, including safe injection kits, fentanyl test strips, and wound care supplies. Vancouver, Canada, deployed a number of “MySafe” machines which use biometric scanning to dispense safe supplies of opioids to prescription holders . AlthougThe objective of the present study was to ascertain the acceptability and feasibility, among different stakeholder groups, of implementing VMHR in Philadelphia. We endeavored to identify barriers that would inform where VMHR should be placed and what harm reduction materials they should contain. This exploratory, multi-stakeholder qualitative inquiry serves as a step toward establishing additional and expanded VMHR in Philadelphia. The study also focuses on contributing to the scientific literature on VMHR and gaining rapid and actionable knowledge about their use, providing a valuable service to our community and public health partners.The University of Pennsylvania and City of Philadelphia Institutional Review Boards approved all procedures. We identified three stakeholder groups to recruit for study participation: (1) end users: individuals served by a Federally Qualified Health Center (FQHC) who have a history of injection drug use and could be potential VMHR users; (2) FQHC staff: patient-facing personnel and administrative leadership and (3) a diverse community stakeholder group that included residents, clergy members, members of community advisory boards and neighborhood organizations. We recruited potential participants from October 2021 to February 2022 through multiple methods including flyer distribution, electronic letters, social media posts, and word of mouth. Interviews were conducted via phone or virtual meeting. Participants were offered a $25 Amazon gift card for participation.In consultation with a local FQHC and the Philadelphia Department of Public Health, we designed a semi-structured interview to address the feasibility and acceptability of VMHR. Working from scripts and an outline, standard definitions of harm reduction and VMHR were provided. The interviewer listed potential materials that VMHRs could dispense: safe disposal and injection kits, fentanyl test sets, items for wound care, naloxone, condoms, and HIV self-tests. Initial questions were open-ended and gave the interviewee an opportunity to describe what they knew about harm reduction, their initial thoughts about a VMHR, and list materials they would like to see in a VMHR, regardless of whether they were relevant to harm reduction. The interviewer then asked about possible challenges to implementing VMHR and how best to introduce VMHR to the community, with specific probes about acceptance, privacy, alternative materials, logistics, and legality. For example, those that may be the potential end users of VMHR were asked how the machine could best serve them, whereas the FQHC staff and leadership interviews focused on the clinical and practical implications of implementing VMHR in their organization. Community members were asked about the potential acceptability of VMHR in their neighborhood and how to introduce the machines to their community. Interviews were digitally recorded, professionally transcribed, anonymized, and lasted from 22-46 minutes. The interview guide is available in Additional file Analysis was guided by modified grounded theory, which provides an adaptive approach to collecting and analyzing qualitative data and has produced robust theoretical models of social behavior in healthcare settings . Following the process outlined by Charmaz and Belgrave , we firsWe interviewed 31 individuals across the four stakeholder groups. The eight potential end users included individuals living with HIV and/or viral hepatitis, individuals who reported current or past injection drug use, and unhoused individuals. From the FQHC, we interviewed nine staff  and six leaders . The eight community members comprised a diverse group and included individuals from multiple zip codes in Philadelphia County. We held interviews with three community advisory board members, two leaders of a faith-based organization, and three residents. Self-reported demographic characteristics are provided in Table Every participant received a standard definition of VMHR “as a machine containing harm reduction materials, such as safe disposal and injection kits, fentanyl test strips, items for wound care, naloxone, condoms, and HIV-self tests.” Stakeholders provided their opinion on these example items and proposed additional items that they believed should be housed in a VMHR. In total, suggested materials spanned five broad categories: (1) supplies to reduce drug use harm , (2) supplies to reduce risk of viral and disease transmission , (3) family planning and feminine products , (4) general medicine and first aid  and (5) informational materials  to connect people to services  or education about substance use disorders, intimate partner violence, and how to use dispensed materials .I would also assume we would want to stuff prevention messages into anything … You know, a little card like, ‘Need help? This is who you call.” …something little, but you know you don’t ever want to miss an opportunity.Stakeholders were generally positive about the idea of a machine that dispensed harm reduction materials and could potentially save lives. Naloxone garnered universal support and was also seen as acceptable to others. Stakeholders were enthusiastic about wound care and first aid equipment. Participants in all stakeholder groups mentioned the importance and benefit of information materials. As one community member stated:The machines themselves sort of share information with patients, right? So, like I would love that even if you’re giving them a needle, you’re also giving them a ‘Here’s some Suboxone places.’Similarly, an FQHC staff member shared:Fentanyl test strips were also viewed positively across stakeholder groups. One end user said that the inclusion of fentanyl test strips and naloxone, “would be a really positive thing,” for his area which had seen a recent increase in opioid use.Like, we know syringe sites work, we know that. So, I think the next best thing because you can’t really have them would be a vending machine where it’s accessible for people. Like if it’s there, people will take it.In contrast, interviewees held conflicting views on the feasibility and acceptability of including safe injection or smoking kits, clean needles, and MOUD. Syringes proved to be the most controversial. In general, FQHC members offered the greatest support, with their chief concern being proper disposal. Community members also supported the provision of clean syringes and saw VMHR as an opportunity to deliver aspects of a safe injection site or syringe exchange site in another, more discreet form. As one community member described:Uh… [I would] not really [be okay with needles being the machine]. I have to be honest- nah, because that’s just giving them a way to get high. It’s easy access because instead of going to the store, and they’re [going to be] selling them or stealing them...Individuals in the end user group similarly demonstrated some support but included a number of interviewees that were against the inclusion of clean needles because they thought it would promote drug use and lead to more litter. One end user expressed reluctance:Yeah, providing Suboxone would present a lot of challenges in terms of safety and making sure that folks don’t prioritize vandalizing the machines in order to steal the Suboxone. You probably would deal with issues concerning the [Drug Enforcement Agency].Opinions on MOUDs were divided within stakeholder groups. One FQHC staff member was in favor of a machine that contains Suboxone  to prevent relapse in emergency situations when an individual’s dose is lost. However, another FQHC staff member noted the complexity of providing such a controlled medication through a machine:That’s why I said if they’re put in areas where you would not expect to see them – City Hall [for example] – I think then people aren’t going to mind seeing them pop up in areas where they’re needed also.Participants discussed the location of the machine at length, including views of desirable or undesirable geographic areas. Almost all interviewees, regardless of stakeholder type, suggested Kensington (an area in Philadelphia with the greatest drug activity), as the most appropriate location for the machine. For example, one community member explained: “Where are they going to make the most impact? Where are they going to meet the most people who are doing this? I would say is obviously Kensington.” However, a number of participants suggested areas outside of Kensington to target neighborhoods with less visible drug use and provide harm reduction materials to underserved communities. One community member advocated for an intentionally conspicuous placement to potentially reduce stigma and normalize the presence of VMHR:Despite the potential advantages of the machines being visible, many interviewees agreed that this should be balanced with protecting the user’s privacy and confidentiality. Nevertheless, many participants expressed that privacy concerns would be fewer in areas with visible open air drug use, like Kensington. One end user was not concerned for user privacy because, “they’re out on the streets where I’m from – I’m from Kensington – they’re out on the street, they don’t care.”Participants also discussed where the machines should be placed in relation to existing buildings and landmarks. Across the three stakeholder groups, stakeholders recommended a variety of places, including public transit infrastructure, recovery centers, libraries, gas stations, grocery stores, schools, and police stations. Stakeholders most commonly endorsed placing VMHR in and around subway stations, particularly in stations close to Kensington. FQHC leadership and staff were generally receptive to the idea of operating a VMHR at their center. However, both administrators and clinicians raised a variety of concerns related to how VMHR would affect patient care, privacy, and willingness to access the FQHC. Although FQHC staff recognized the potential for VMHR to serve individuals at all hours, at least one staff member thought that VMHR may be more helpful in other locations given that the FQHC already provides harm reduction materials directly.[VMHR] being in the bathroom might encourage [drug] use in the bathroom as well. So, they may not want to do it in the bathroom, but it has to be in a place where people don’t feel like everybody’s watching them take items from the machine or get items dispensed from the machine.Most stakeholders addressed the pros and cons of different placements. Stakeholders viewed an attractive location for VMHR as one that balanced visibility, security, and privacy. Visibility was seen as increasing security and accessibility, and reducing stigma, but was potentially concerning due to end user’s privacy concerns and community opposition. In general, FQHC staff were more sympathetic to privacy concerns than the other groups. For instance, one staff member conceded that individuals may be more comfortable accessing fentanyl strips through a machine than asking their treatment provider. Yet, other FQHC staff were concerned that their patients would access the machine in lieu of seeing a treatment provider, reducing clinician and patient interaction and engagement. Interviewees discussed the nuances of placing the machine in other settings, such as whether it was prudent to place it in a waiting room given the lack of confidentiality or a bathroom given the lack of supervision. As one end user described,Finally, some interviewees suggested placements that would preserve 24/7 access to VMHR as well as decrease vandalism and theft. For example, several interviewees suggested small, kiosk-like housing, comparable to banks that have ATMs in their entryways.How would someone pay since this is a marginalized community we’re talking about, and income is very low, how would they be able to afford to get something out of those machines?Stakeholders mentioned a variety of considerations for how the machine should be accessed and under what conditions. There was disagreement about whether items should be free. FQHC staff often noted that cost could be prohibitive for many individuals who could be served by the machine:…the way I look at it- if everything in it is ‘free,’ I really think that one person’s going to go back because, ‘they will let me take 10 of these or 12 of these’ or keep taking them until there’s none left.Several individuals with lived experience pointed out that some items (such as syringes) can be obtained freely elsewhere so, “they're going to go where they can get them for free.” On the other hand, many participants reported misgivings about free items, commonly expressing concerns that the machine would be “cleaned out”. One community member explained:if the access to the materials they’re contained in is free, then I would almost think you could legally view that as an inducement to get high.There were also ideological concerns about the optics of free items as “condoning drug use.” As one end user cautioned:Some interviewees recommended imposing more conditions of access to have more control over the machine’s users, such as restricting access to children, limiting access to certain times of day, or surveilling users more closely. Yet, stakeholders expressed competing concerns of balancing surveillance for security with privacy and accessibility for users (particularly for machines outside of highly visible drug activity areas). Stakeholders brought up related concerns that a machine would attract law enforcement that would turn potential users away or criminalize users for accessing or obtaining drug paraphernalia. Some FQHC staff members recognized the clinical utility of collecting data on what sort of materials were being accessed. One staff member mused:I would say it would have to be somewhere linked to us and that someone would actually have to monitor how many times that person has used the vending machine within a day, within week and what exactly that they’re, you know getting to, right?Many participants offered alternative methods of accessing items, such as a machine-specific access card, allowing access through a Medicare or SNAP benefits card, non-money token, or user-specific codes.Some interviewees acknowledged that the accessibility and anonymity of the machine may depend on the materials. All stakeholders who discussed naloxone agreed that it should be dispensed freely and quickly in case of an emergency.We asked stakeholders about the community response to VMHR and how to improve community acceptance. Interviewees generally assumed that there would be dialog between the implementers of the machine and the community in which they would be placed. Stakeholders offered a variety of strategies toward how best to engage the community around VMHR, including group-based or one-on-one educational meetings and open forums addressing concerns, relationship building with neighborhood leaders or community organizations, and advertising through flyer distribution and social media. Stakeholders highlighted that coupling VMHR with safe and increased disposal of needles would be a particularly important selling point to the community. A number of interviewees recommended starting with less stigmatized materials  and gradually introducing more controversial materials . Other features that would enhance acceptability of machines include limiting children’s access, and VMHR attendants such as peer specialists or guards to interface, educate, and assist. FQHC employees and community stakeholders promoted education around harm reduction efforts in general, to help the community to understand, “that ultimately the community is protected through these measures.”Because I’m a recovering heroin addict and I feel like if it was easier for me to get syringes and things like of that nature, then I might not have stopped using drugs.Interviewees suggested reasons why some community members might be against the machines. One explanation was that the VMHR (providing items such as safe injection kits) condones and encourages drug use. End users were the only stakeholder group to note that they themselves held this belief. One end user in recovery from OUD described his opposition to syringes:If I’m driving down the street and I see one of those machines, does that immediately label that neighborhood as a more drug-prone neighborhood?Another proposed reason for community opposition was what the machine signified about the neighborhood and that the machine might attract increased drug activity, drug litter, and drug-using individuals. As one community member explained:I think in different neighborhoods where there was discussion about potentially putting safe injection facilities, there was a lot of uproar and so, I don’t know whether you would get the same uproar putting something like a vending machine in the same areas.Participants from all stakeholder groups drew comparisons with the city’s recent initiative to introduce safe injection sites and reflected on lessons learned. As one FQHC employee noted:We have to start thinking radically about what to do with this crisis…we definitely need some radical ideas and folks need to embrace that we need some radical ideas to stop people from dying.Many stakeholders believed the main lesson learned from the failed safe injection site initiative was a lack of communication between the implementers and the community. Despite this, most stakeholders were hopeful that with a thoughtful introduction coupled with significant community outreach, discourse, and education, the community would be receptive to VMHR due to the salience and prevalence of drug use in many neighborhoods. Some community members highlighted that the need for VMHR was pressing given the severity of the opioid overdose epidemic and that universal community acceptance was neither necessary nor possible. As one community member described:Our study identifies critical decision points relating to the feasibility and acceptability of VMHR in Philadelphia. There was striking consensus across many domains among our stakeholder groups. For example, there was universal agreement on providing naloxone, wound care and first aid materials, and informational materials to connect individuals to services and treatment. The agreement about these harm reduction materials may indicate that public attitudes are shifting in favor of certain harm reduction interventions for PWID. No stakeholders mentioned a moral hazard of providing naloxone or provided other ideological debates that naloxone promotes or encourages drug us , 38. StaConsistent with previous research, there was less agreement among stakeholder groups about providing syringes or clean needles, with end users holding the most negative views. A central finding of our study was the perception that VMHR or certain materials  promote or encourage drug use. While end users were the only group to personally subscribe to this idea, all stakeholder groups noted the stigma against harm reduction interventions as a primary reason for community opposition. This is a critical opportunity for broad public health messaging about harm reduction, as well as for harm reductionists to learn how to frame harm reduction in ways that are persuasive to the community and particularly to people who use or inject drugs. There is a body of research that contradicts that harm reduction interventions promote substance use. This evidence supports the reverse, that harm reduction interventions have been linked to positive health outcomes for PWID both in the short term, and in the long term by preventing infectious diseases and connecting people to treatment , 22, 31,Participant stakeholders had many suggestions for increasing community and neighborhood acceptance of VMHR. While we directly asked how to enhance community acceptance of VMHR, Philadelphia’s recent failure to implement a safe injection facility following intense community backlash meant that this topic was on participants’ minds. Many stakeholders compared the approach of the safe injection site introduction and provided different strategies for introducing a VMHR. Education and small community meetings were the most recommended strategies. Stakeholders agree that these machines should be placed in areas of highest drug use and need. Our findings also indicate that a VMHR becomes a symbol or a marker for what and who a community is. For example, a number of community members and end users believed that a VMHR would identify a neighborhood as one with illicit drug use , or evenStakeholders converged on a common set of ideas to make the machine more acceptable: link it to increased needle disposal in the neighborhood and include informational materials connecting people to services and treatment. There are many research and evaluation opportunities to test if or what kind of messaging effectively connects individuals to treatment or other harm reduction services. One proximal impact of the informational materials is that their inclusion increases community enthusiasm for the VMHR. The present findings suggest that VMHR could be made more palatable by including these materials. In general, stakeholders suggested a variety of “foot-in-the-door” approaches to introducing VMHR to the community, which is promising given a VMHR that dispenses naloxone is already in operation in Philadelphia , 40.ThesSeveral study limitations should be noted. First, the sample represents a regionally confined subset of stakeholders who responded to a recruitment letter about a VMHR. It is possible that individuals who agreed to participate are not representative of the larger Philadelphia population and that a more diverse sample would show different outcomes. Second, we did not interview any legal professionals. Although several individuals mentioned critical legalities of providing certain materials . Future inquiries should seek to understand the multi-level interactions between local, state, and federal laws.A suite of public health interventions is necessary to respond to the complex interaction of the opioid and HIV epidemics, the ongoing COVID-19 pandemic, and the unique social and political contexts of diverse urban environments. This study is one of the first to investigate the potential feasibility and acceptability of an innovative modality for delivering harm reduction materials in the USA. Stakeholders were generally positive about a VMHR and provided a number of suggestions to increase community acceptance around this strategy. Given the growing number of VMHR in the USA, more research and evaluation are needed to examine community acceptance of VMHR, and evaluate VMHR effectiveness in increasing access and acceptability of harm reduction supplies. Further, it will be important to learn if VMHR could successfully link individuals to treatment and to explore local potential negative effects of VMHR  that were suggested by stakeholders.Additional file 1. Interview script and question guide.


Acacia nilotica methanolic extract. The phytochemicals were detected using gas chromatography–mass spectrometry (GC–MS) while the in vivo antidiarrheal test was done using Swiss albino mice. To determine the details of the mechanism(s) involved in the antispasmodic effect, isolated rat ileum was chosen using different ex vivo assays by maintaining a physiological environment. GC–MS results showed that A. nilotica contained pyrogallol as the major polyphenol present (64.04%) in addition to polysaccharides, polyphenol, amino acid, steroids, fatty acid esters, and triterpenoids. In the antidiarrheal experiment, A. nilotica inhibited diarrheal episodes in mice significantly (p < 0.05) by 40% protection of mice at 200 mg/kg, while 80% protection was observed at 400 mg/kg by the orally administered extract. The highest antidiarrheal effect was observed with loperamide (p < 0.01), used as a control drug. In the ex vivo experiments, A. nilotica inhibited completely in increasing concentrations (0.3 to 10 mg/mL) the carbachol  and high K+ (80 mM)-evoked spasms in ileum tissues at equal potencies (p > 0.05), similar to papaverine, a dual inhibitor of the phosphodiesterase enzyme (PDE) and Ca++ channels. The dual inhibitory-like effects of A. nilotica on PDE and Ca++ were further validated when A. nilotica extract (1 and 3 mg/mL)-pre-incubated ileum tissues potentiated and shifted isoprenaline relaxation curves towards lower doses (leftward), similar to papaverine, thus confirming the PDE inhibitory-like mechanism whereas its CCB-like effect of the extract was confirmed at 3 and 5 mg/mL by non-specific inhibition of CaCl2-mediated concentration response curves towards the right with suppression of the maximum peaks, similar to verapamil, used as standard CCB. Thus, this study characterized the chemical composition and provides mechanistic support for medicinal use of A. nilotica in diarrheal and hyperactive gut motility disorders.This present study evaluated and rationalized the medicinal use of the fruit part of  Diarrhea, characterized by an increased frequency of bowel movements, wet stool, and abdominal cramps, is a serious health problem ,2. Diarr++ . Thus, anditions .Currently available treatments for diarrhea are non-specific and generally, drugs are used to reduce the uneasiness and discomfort of recurrent bowel movements . AvailabAcacia nilotica (L.) Wild ex. Del., commonly known as Mimosa nilotica (family Mimosaceae), is a medium-sized tree that is known locally as ‘Babul’ or ‘Kikar’ [A. nilotica [A. arabica, A. abyssinica, and A. seyal, are used in traditional medicine to treat leprosy, tuberculosis, skin ulcers, dysentery, cough, smallpox, toothache, and malignancies, as well as used as astringents, antispasmodics, antidysentrics, and aphrodisiacs [A. nilotica is a possible source of antioxidant polyphenols [ ‘Kikar’ . Africa,nilotica . Other Aodisiacs ,19,20. Podisiacs  and are odisiacs . In receodisiacs ,24,25. Podisiacs . A. niloyphenols ,28,29, aA. nilotica, the plant applied in this research, is used in local folk medicine to treat a range of ailments, there is no solid scientific data to support the use of A. nilotica fruit extract in the treatment of diarrhea. As a result, the aim of the work was to use in vivo and ex vivo experiments for phytochemical investigation using GC–MS as well as to discover the exact mechanism(s) implicated in the putative gastrointestinal inhibitory effects of A. nilotica fruit extract.Although A. nilotica from a local market in Dammam (Saudi Arabia), it was identified and authenticated using macroscopic and microscopical examination by Dr. Abuzer Ali, Department of Pharmacognosy, Taif University, Taif Saudi Arabia and matched with a Pharmacopoeial standard. . The plant sample was preserved at the herbarium with the voucher # PL/0445/2020-21/P-008. With the use of a mixer grinder, the plant material was powdered; 40 g of powdered crude sample was placed in a Soxhlet device and extracted with 200 mL of methanol at 70 degrees Celsius. The extract was concentrated using a rotary evaporator after rigorous extraction . For future investigation, the concentrated extract was maintained in an airtight glass jar at 5–10 °C. The extract was GC–MS examined using earlier reported methods [After purchasing the fruit of  methods ,31.Sigma provided carbamylcholine (CCh), papaverine, isoprenaline, Ethylenediaminetetraacetic Acid (EDTA), verapamil, acetylcholine perchlorate (ACh), and loperamide . To make the physiological buffer solution (Tyrode), the following reagents  were used: magnesium sulphate, potassium chloride, glucose, potassium dihydrogen phosphate, calcium chloride, sodium chloride, and sodium bicarbonate . All of the substances were of analytical quality, except castor oil acquired from a local drugstore.From the Animal Care Unit, ‘College of Pharmacy, Prince Sattam bin Abdulaziz University, Saudi Arabia’, Swiss albino mice (25–30 g) were obtained for in vivo studies and rats (200–250 g) for ex vivo experiments and were kept at a temperature optimum (22 °C), relative humidity (55%), and exposure to a light/dark cycle. All animals were fed a regular diet of pellets and had unrestricted access to water. Prior to the ex vivo studies, mice fasted for 24 h, and cervical dislocation was performed under light sedation, with death confirmed by elimination of ear reflexes. All experiments (in vivo and ex vivo) were carried out with caution and in accordance with the guidelines outlined in the NRC . The BioA. nilotica was per-formed by GC–MS to detect the presence of several phytoconstituents. The chromatographic separation of metabolites was carried on a capillary column 60 M TRX 5-MS (30 m × 250 µm I.D. 0.25 µm film) using 2 μL of sample injection volume. The oven temperature program was as follows: 80 °C initially for 3 min and then ramped at a rate of 10 °C/min to 280 °C for 19 min. The carrier gas was set at a constant flow rate of 1.21 mL/min. The injection port, transfer line, and ion source were set to 260 °C, and the mass-scanning range was set to 40 to 650 m/z in scan mode. The injection was executed in split mode with a 10:1 split ratio, and a 3-min solvent delay time was set for the samples. Identification of individual phytoconstituents was achieved using National Institute of Standards and Technology (NIST) libraries and the mass spectra of literature [The phytochemical investigation of the methanolic extract of terature .A. nilotica methanolic extract, 200 and 400 mg/kg, respectively, after a pilot screening for dose selection. As a positive control, the fourth group of mice was administered loperamide (10 mg/kg). Each animal was kept in the cage, with a blotting sheet on the floor to allow a blind observer to determine the presence or absence of diarrhea. All mice were given castor oil (10 mL/kg) orally after an hour. All blotting sheets from individual cages were checked for typical diarrheal droppings after 4 h. If no diarrheal spots were noticed on the blotting sheet, protection was documented [Twenty mice were divided into five groups, each with an equal number of mice, at random. Mice in the first group were administered an oral gavage of saline (10 mL/kg) after a twenty-four-hour fast and were labeled as the negative control group. The second and third groups (test groups) were given two increasing doses of cumented ,35.2, 11.90 NaHCO3, 0.42 NaH2PO4, 1.8 CaCl2, and 5.55 glucose, (pH 7.4) The tissues were stabilized for 30 min with the addition of acetylcholine (0.3 M) at regular intervals (5 min) while 1 g tension was applied by clockwise rotation of the transducer knob. CCh and high K+ (80 mM) were employed to induce prolonged contractions after stabilization, and A. nilotica was added to the bath solution in increasing concentrations until the maximal and/or complete relaxation of tissue was achieved. The inhibitory effect of A. nilotica on CCh and K+-mediated contractions was observed, which could indicate pharmacodynamics such as voltage-gated Ca++ channel blockade and/or PDE inhibition. Multiple smooth muscles are depolarized by K+ (>30 mM), which activates Ca++ channels (L-type), resulting in prolonged contractions [+-mediated contractions, whereas verapamil (CCB) shows significantly higher potency against high K+ compared to CCh-mediated contractions [A previously documented approach was used to sacrifice rats and to separate the final part of the small intestine (ileum) . Ileum tractions . PDE-inhractions .A. nilotica against high K+, ileum tissues were incubated in Ca++-free Tyrode’s solution with EDTA (0.1 mM) for 45 min to confirm Ca++ channel blocking (CCB). A Ca++-free solution was replaced with a K+-rich and Ca++-free Tyrode’s solution at the following concentrations (mM): KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.90, NaH2PO4 0.42, glucose 5.55, and EDTA 0.1. After 45 min of incubation in this solution in the presence and absence of increasing concentrations of A. nilotica, CaCl2 CRCs were produced, and the findings were compared to the standard CCB agent, verapamil [After the observation of preliminary relaxation of erapamil .A. nilotica against high K+ and CCh at identical concentrations is an indication of PDE inhibition [A. nilotica were used to indirectly validate PDE inhibition. PDE blockage was indicated by the potentiation of isoprenaline curves to the left, similar to papaverine, a typical PDE inhibitor, utilized as a control [The relaxing effect of hibition ; therefo control .n” being the number of experiments that were repeated. The median effective concentrations (EC50) are geometric means with 95% confidence intervals (CIs). The statistical criteria utilized for multiple comparisons of concentration–response curves (CRCs) with controls were Student’s t-test or two-way ANOVA followed by Bonferroni’s post-test. W the Chi-square (χ2) test, all groups were statistically compared to a saline control group for diarrhea protection. p < 0.05 was regarded as statistically significant. For CRC regression analysis, Graph Pad Prism (version 4) was used.The statistical analyses were performed as the mean ± standard error of the mean (SEM), with “A. nilotica yielded 36.47% of methanolic crude extract.The fruits of A. nilotica methanolic extract revealed the presence of 19 phytoconstituents representing 99.03% that were identified by compairing with mass spectrum library of NIST. All separated phytoconstituents, peak area, % area, retention index, and molecular formula with the chemical structure of A. nilotica are shown in A. nilotica. These phytoconstituents were tentatively identified by compairing their mass spectra with the NIST library  and 5.84 mg/mL , respectively. Papaverine had similar relaxing effects on CCh and high K+-induced spasms, with EC50 values of 9.82 M  and 9.24 M , respectively  and 2.82 M , respectively.As demonstrated in ectively B. As demA. nilotica (1 and 3 mg/mL) confirmed PDE inhibitory activity by shifting the isoprenaline-induced inhibitory CRCs to the left . Similarly, papaverine, a dual Ca++ channel and PDE inhibitor [+-evoked spasms at comparable concentrations, but verapamil, a typical CCB [+ at a lower concentration compared to CCh. This indicates that, similar to papaverine, A. nilotica has dual inhibitory mechanisms for PDE inhibition and Ca++ channels. PDE-inhibitors, which block PDE, result in a cAMP increase in tissues and thus cause relaxation. PDE hinders smooth muscle relaxation by converting cAMP into its inactive form (AMP) [A. nilotica was evaluated indirectly for PDE-inhibition and cAMP elevation by constructing isoprenaline-induced inhibitory CRCs in the absence and presence of pre-incubated tissues with the test substance. In pre-incubated ileum tissues of A. nilotica, potentiation of isoprenaline’s inhibitory CRCs towards lower dosages (leftward) verified its PDE-inhibitory character, and the results were equivalent to papaverine, a known PDE-inhibitor [A. nilotica extract, it was tested for Ca++ ion inhibitory effect.To provide the basis to the traditional medicinal report of t spasms , the methe colon . Similarhe colon , pre-admactivity . Based oblocking  and/or Pblocking , we eval high K+ . The EC5nhibitor , suppresical CCB ,48, selerm (AMP) . Hence, nhibitor . In orde+ (˃30 mM)-mediated spasm are considered as CCBs [A. nilotica, in previously Ca++-free tissues, the ileum tissues were preincubated with A. nilotica at increasing concentrations. Ca++-CRCs were made in the absence of A. nilotica and pre-incubated tissues with A. nilotica, which repelled Ca++-CRCs to the right with suppression of the maximum peak, similar to papaverine, a dual inhibitor of PDE and Ca++ channels. The plant Ca++-CRC comparison with verapamil, a standard CCB [A. nilotica. Previously published findings of the antispasmodic effect of A. nilotica pods in rabbit jejunum support this CCB-like effect [A. nilotica methanolic extract. Pyrogallol was discovered to be one of the major phytoconstituents of A. nilotica; it is a polyphenol that is present in (64.04%) the extract and has antibacterial activity [Substances that reverse high K as CCBs , hence tdard CCB , furthere effect . Polysacactivity , whereasactivity . In COPDA. nilotica and indicates pyrogallol as the major polyphenol present in addition to the polysaccharide, polyphenol, amino acid, steroids, fatty acid esters, and triterpenoids. The in vivo antidiarrheal and ex vivo antispasmodic assays conducted in rodents indicate that A. nilotica possesses dose-mediated protection in mice from castor-oil induced diarrhea similar to loperamide while its preincubation in isolated rat ileum potentiated the isoprenaline-mediated inhibitory curves whereas the Ca++ CRCs were shifted towards right with suppression of the maximum response, thus confirming its antispasmodic effect possibly mediated by a combination of PDE-inhibition and Ca++ channels antagonist-like mechanisms, though additional mechanism(s) cannot be ignored.These findings characterized the chemical composition of the methanolic extract of


Our methodology involved the protonation of samin (5) to generate an oxocarbenium ion followed by the attack from two different nucleophiles, namely, thiols (RSH) and alcohols (ROH). The highly diastereoselective thioether and ether furofuran lignans were obtained, and their configurations were confirmed by 2D NMR and X-ray crystallography. The mechanism underlying the reaction was studied by monitoring 1H NMR and computational calculations, that is, the diastereomeric α- and β-products were equally formed through the SN1-like mechanism, while the β-product was gradually transformed via an SN2-like mechanism to the α-congener in the late step. Upon evaluation of the inhibitory effect of the synthesized lignans against α-glucosidases and free radicals, the lignans 7f and 7o of the phenolic hydroxyl group were the most potent inhibitors. Additionally, the mechanisms underlying the α-glucosidase inhibition of 7f and 7o were verified to be of a mixed manner and noncompetitive inhibition, respectively. The results indicated that both 7f and 7o possessed promising antidiabetic activity, while simultaneously inhibiting α-glucosidases and free radicals.The transformation of sesame lignans is interesting because the derived products possess enhanced bioactivity and a wide range of potential applications. In this study, the semisynthesis of 28 furofuran lignans using samin ( Numerous furofuran lignans have been isolated from various plants, and their biological activities have been evaluated; for example, antitumoral activity of syringaresinol  [1], choiver  × 100, where A1 and A0 are the absorbance with and without the sample, respectively. The IC50 value was deduced from a plot of percentage inhibition versus sample concentration and acarbose was used as a positive control. The experiment was performed in triplicate.The α-glucosidase inhibitory activity against rat intestinal maltase and sucrase was determined according to our previous report . The crui and Ki′ values, the slope and intercept from the Lineweaver–Burk plot were replotted vs. [I], which provided the secondary plot.For kinetic analysis of the active compound, α-glucosidases and active compounds were incubated with increasing concentrations of maltose (0.5–8 mM) and sucrose (5–80 mM). The type of inhibition was determined by the Lineweaver–Burk plot. For calculation of K0 − A1)/A0] × 100, where A0 is the absorbance without the sample, and A1 is the absorbance with the sample. The SC50 value was deduced from a plot of percentage inhibition versus sample concentration. Butylated hydroxytoluene (BHT) was used as the standard control and the experiment was performed in triplicate.The radical scavenging activity was validated using the DPPH colorimetric method. Briefly, the synthesized compounds (20 μL) were added to 0.1 mM methanolic solution of DPPH (100 μL). The mixture was kept in the dark at room temperature in an incubator shaker for 15 min. The absorbance of the resulting solution was measured at 517 nm with a 96-well microplate reader. The percentage inhibition was calculated by  × 100, where A0 is the absorbance without the sample, and A1 is the absorbance with the sample. The SC50 value was determined from a plot of percentage inhibition versus sample concentration. Butylated hydroxytoluene (BHT) was used as the standard control and the experiment was performed in triplicate.The radical scavenging activity of synthesized compounds against ABTS•+ was carried out according to a procedure described previously . Briefly2α-7f and on a Bruker X8 APEX II Kappa CCD diffractometer using graphite monochromatized Mo-Ká radiation (ë = 0.71073 Å) for 2β-7fBr. The structures were solved using SHELXTL XT 2013/1 [2α-7f and 2β-7fBr is given in Single crystal X-ray diffraction data were collected at 296(2) K on a Bruker X8 Prospector Kappa CCD diffractometer using an IìS X-ray microfocus source with multilayer mirrors, yielding intense monochromatic Cu-Ká radiation ë = 1.54178 Å) for T 2013/1 , expande8 Å for TT 2013/1  that areComplete geometry optimization was carried out with the density functional theory (DFT) calculations using the popular hybrid method (B3LYP) with the 6-31 + G basis set. The methanol phase and the polarizable continuum model (PCM) calculations were performed using the Gaussian09 package   with def5) and nucleophiles  under acidic conditions. This synthetic approach provided 28 new furofuran lignans. Additionally, ether furofuran lignans displayed a diastereoselectivity higher than that of the thioether furofuran lignans. All synthesized compounds were fully characterized via different spectroscopic techniques (HRESI-MS and NMR) and single crystal X-ray diffraction. The reaction mechanisms of samin and alcohol nucleophiles were investigated for the first time. The NMR monitoring and DFT calculations suggested that the products were presumably formed through the SN1-like mechanism by protonation of the hemiacetal center of samin (5) to generate the corresponding oxocarbenium ion as the intermediate. The subsequent reaction of this oxocarbenium ion with the alcohols then led to the observed only α-form either directly, or alternatively, by protonation of their epimer (β-form) through the SN2-like transition state contributed by acid-catalysis. Furofuran lignans 2α-7f and 2α-7o, with a phenolic hydroxyl group, displayed remarkable inhibitory activities against rat intestinal maltase and sucrase as well as free radicals (DPPH and ABTS). Furthermore, the active 2α-7f and 2α-7o were selected as representatives for the kinetic analysis. The kinetic results revealed that 2α-7f and 2α-7o inhibited maltase and sucrase by mixed-type and noncompetitive inhibition. The present investigation provides fundamental clues to the structural motifs required for synthesizing a new series of improved inhibitors and a practical synthetic approach. In conclusion, a new series of thioether and ether furofuran lignans were obtained by nucleophilic substitution between starting samin (


Artemisia annua L. has long been known for its medicinal properties and isolation of ingredients whose derivatives are used for therapeutic purposes. The CYP2B6 and CYP3A4 enzymes belong to a large family of cytochrome P450 enzymes. These enzymes are involved in the metabolism of drugs and other xeonobiotics. It is known that various compounds can induce or inhibit the activity of these enzymes. The aim of this study was to investigate the nature of the inhibitory effect of Artemisia annua extract on CYP2B6 and CYP3A4 enzymes, as well as the type of inhibition, the presence of reversible or pseudo-irreversible inhibition, and the possible heme destruction. The methanolic extract of Artemisia annua showed an inhibitory effect on CYP2B6  and CYP3A4 enzymes . A significant decrease in heme concentration by 46.8% and 38.2% was observed in different assays. These results clearly indicate that the studied plant extracts significantly inhibited the activity of CYP2B6 and CYP3A4 enzymes. Moreover, they showed irreversible inhibition, which is even more important for possible interactions with drugs and dietary supplements. Artemisia annua L. (sweet wormwood) continue to engage and fascinate scientists around the world. Artemisia annua  enzymes are a large family of enzymes found in all biological kingdoms, including humans. In humans, the CYP enzymes are located in the mitochondria or endoplasmic reticulum. This large group of enzymes are monooxygenases; they require an external oxygen donor to function properly. According to their structure, CYP enzymes are hemoproteins; they have heme as a prosthetic group . CYP enzN-demethylation of S-mephenytoin. Efavirenz, which converts CYP2B6 to hydroxylated metabolites, is also used for this purpose [The CYP2B6 enzyme is expressed in the liver and to some extent in the lungs . It is a purpose . Other s purpose ,19. Besi purpose ,20.CYP3A4 is the most important drug-metabolizing enzyme in the CYP group, as it metabolizes about 33% of drugs. It is an enzyme responsible for 20% of all reduction and oxidation reactions within the CYP group . Since iArtemisia annua L. extract (AAE) on the activity of CYP2B6 and CYP3A4 enzymes, as well as to examine heme binding and pseudo-irreversible and reversible inhibition.The aim of this study was to investigate the inhibitory effect of Artemisia annua L. plant were purchased on the market . Cytochromes P450 2B6 and 3A4 (recombinant) were co-expressed with nicotinamide-adenine-dinucleotide phosphate (NADPH) reductase and obtained from Thermo Fisher Scientific , as well as cytochrome b5 in baculosomes. The contents of CYP2B6 and CYP3A4 were confirmed to be 1 μM, as was declared by the manufacturer, based on the cytochrome P450 carbon monoxide assay [+), glucose-6-phosphate (G6P), and glucose-6-phosphate dehydrogenase (G6PDH) obtained from Sigma-Aldrich  were used to prepare the generation system. Formic acid  was purchased from Semikem , potassium phosphate (p.a.) and dichloromethane (p.a.) from Kemika , methanol from Merck KGaA , ultrapure water from Sigma-Aldrich, and acetonitrile from KEFO . In order to prepare a potassium phosphate buffer of pH 7.4, a potassium dihydrogen phosphate obtained from Kemika d.d. was used. Sodium hydroxide (Semikem d.o.o.) was used to adjust the pH. For the marker reactions for CYP2B6 and CYP3A4, S-mephenytoin, S-nirvanol, testosterone, and 6β-hydroxytestosterone from Sigma Aldrich were used. Clopidogrel and troleandomycin, used as positive controls for enzyme activities, were obtained from the Agency for Medicines and Medical Devices of Bosnia and Herzegovina . Pyridine (p.a.) (Semikem d.o.o.), bovine hemin (Sigma-Aldrich), and dimethylsulfoxide (DMSO) (Semikem d.o.o.) were used in the hemochromopyridine assay. Potassium hexacyanoferrate (PCF)  and diltiazem  were used in the study of reversible and pseudo-reversible inhibition. Plant extracts were shaken on an orbital shaker with mat platform (Sigma-Aldrich). Superoxide dismutase (SOD) (Sigma-Aldrich), catalase (CAT) (Sigma-Aldrich), and hydrogen peroxide  (Semikem) were used to test out the binding specificity of the enzyme. A water bath (Thermo Fisher Scientific) was used for enzyme incubations. Samples were centrifuged using the FC5306 mini centrifuge . Assay of residual enzyme activity was performed using high-performance liquid chromatography coupled with UV-Vis detection . To record the sample spectra, a spectrophotometer UV-1280  was used. In order to retrieve the enzyme activity, Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific) were used.The aerial parts of the de assay . β-nicotArtemisia annua (AAE) was obtained according to the method described by Mashati et al. [The extract of i et al. . The aer+:1000 IU/mL G6PDH = 50:25:1 (v/v/v) was prepared immediately before use. This generating system contains glucose-6-phosphate dehydrogenase, which regenerates NADP+ to NADPH, keeping the concentration of cytochrome P450 co-enzyme (NADPH) constant during incubation. The reaction was started by adding the generating system . In order to determine the enzyme residual activity, S-mephenytoin and testosterone  were used. Then, 1 mL of ice-cold 1% solution of formic acid in dichloromethane was used to terminate the reaction. The samples were mixed and then centrifuged at 1900× g for 10 min. After centrifugation, two layers (water and organic layer) were formed and 850 µL of the organic layer was transferred to cuvettes and evaporated.In order to determine the nature of the inhibitory effect of AAE on CYP2B6 and CYP3A4, three types of inhibition assays were performed: direct, time-dependent, and metabolic inhibition assays of the enzyme. In the direct inhibition assay, no preincubation of AAE with the enzyme was performed. The generating system was added directly to the incubation mixture with the substrate and incubated for 15 min. In the time-dependent inhibition assay, AAE was first pre-incubated with the enzyme in a water bath for 30 min, and then the reaction was started by adding the generating system and substrate. The samples were then incubated for an additional 15 min. In the metabolism-dependent inhibition assay, AAE, enzymes, and the generating system were first pre-incubated for 30 min, and the reaction started by adding the substrate. The samples were incubated for an additional 15 min. Enzyme incubations were performed in triplicate, with mechanical stirring in a water bath at 37 °C. Aliquots of AAE with a final concentration of 10 μg/mL dissolved in methanol were transferred to glass tubes and evaporated to dryness, except for the control samples without the inhibitor (AAE). After evaporation of the solvent, an incubation mixture with a volume of 100 μL was prepared, consisting of 5 pmol CYP2B6 or CYP3A4 enzyme, 50 mM potassium phosphate buffer (pH 7.4), and ultrapure water. An NADPH-generating system composed of 0.1 M G6P:10 mg/mL NADPN-demethylation of S-mephenytoin to S-nirvanol was observed. The mobile phase consisted of acetonitrile and water in a ratio of 40:60, v/v. The analysis was performed isocratically. The flow was set at 1.0 mL min−1. The injection volume was adjusted to 10 µL. Chromatograms were recorded at 210 nm. The duration of the analysis was set to 20 min. The retention time of S-mephenytoin was 12.3 min and that of S-nirvanol was 9.3 min [v/v. The analysis was performed isocratically. The flow was set to 1.0 mL min−1. The injection volume was set at 10 µL. Chromatograms were recorded at 254 nm. The duration of the analysis was set at 35 min. The retention time of testosterone was 19.2 min and that of 6β-hydroxytestosterone was 5.8 min [Next, 30 μL of methanol was used to dissolve the sample, which was analyzed using HPLC Agilent Zorbax SB C18 column  from Agilent Technologies . To test the activity of CYP2B6, a marker reaction of  5.8 min . In bothThe hemochromopyridine experiment was used to determine whether or not heme might be destroyed by reactive intermediates produced by the cytochrome P450 cycle. It was carried out with some changes to the procedure published by Flink and Watson  and PaulS-mephenytoin and testosterone as marker substrates . Since the NADPH-generating system was also dialyzed, it was again added to the incubations to initiate the enzyme reaction. Samples were incubated for 30 min with the same settings. An ice-cold solution of formic acid in dichloromethane was used to terminate the reaction process. HPLC was used to analyze the samples. In the case of CYP3A4, diltiazem was used to test out the pseudo-irreversible inhibition. A full recovery of enzyme activity was observed, and the results were used as positive control.One of the characteristics of the catalytic cycle of cytochrome P450 enzymes is the formation of covalent complexes with the ferrous form of iron. Pseudo-irreversible inhibitors have the possibility of rehabilitating enzyme activity after oxidation of heme iron with the use of oxidants. Therefore, three types of tests were conducted to test the pseudo-irreversible nature of the inhibitor: incubation mixture without AAE (control), incubation mixture with AAE, and incubation mixture with AAE to which an oxidant was added after incubation. Incubations lasted 30 min according to the conditions described earlier. After incubation, samples were dialyzed, and 20 mM potassium hexacyanoferrate was added to certain samples . The casR  and Microsoft Excel . Statistical significance between the samples and controls were tested using the Student’s t-test for the estimation of statistical difference (p < 0.05) and Mann–Whitney U-test was used to test the data normality.All incubations in the assays in this study were done in triplicate. Residual activity calculations and statistical analysis were made using the program Of all the observed types of inhibition, AAE most strongly inhibited the activity of CYP2B6 and CYP3A4 enzymes in the metabolism-dependent inhibition assay . In thisp < 0.05). The time-dependent inhibition assay showed no statistically significant inhibition of the enzymes (p > 0.05).The remaining activity of CYP2B6 was also significantly reduced in direct inhibition assays (49.1% of the remaining enzyme activity), whereas no significant inhibition was observed in the time-dependent inhibition assay (the remaining enzyme activity was 96.4%). The remaining activity of the CYP3A4 enzyme in the direct inhibition assay was also significantly reduced and amounted to 39.7%. No decrease in enzyme activity was observed in the study of time-dependent inhibition of CYP3A4. The remaining activity was 99.9%. Direct inhibition of the enzyme was also pronounced in the direct inhibition assay. Although the remaining activity of CYP2B6 and CYP3A4 enzymes was higher by 28.5% and 20.0%, respectively, the inhibition was still statistically significant . After the addition of SOD and CAT, the heme concentration was 53.2% and 61.8%, respectively, compared to the control (no AAE). These results confirm that the destruction in heme concentration is due to reactive metabolites in AAE.The tests were confirmed by the addition of SOD and CAT. A statistically significant decrease in heme concentration was observed in both cases (p < 0.05)  .p > 0.05). No reversible or pseudo-irreversible inhibition was observed.The remaining activity of the CYP2B6 enzyme after incubation with AAE and dialysis was 20.3%, and the remaining activity of CYP3A4 was 20.9%. After dialysis and addition of PCF, the remaining activity of CYP2B6 and CYP3A4 was 38.4% and 36.9%, respectively. Reversible inhibition is characterized by the return of activity after dialysis. Pseudo-irreversible inhibition is characterized by the return of activity after dialysis and addition of an oxidant. In this case, no statistically significant difference was found between the two observed groups  and CYP3A4 (IC50 = 4.93 µM) enzyme activity. In addition, this study compared the effect of Artemisia annua leaves in the form of tea on the activity of the above enzymes. The inhibition of the enzymes was also observed, namely IC50 = 2.31 µM for CYP2B6 and IC50 = 5.67 µM for the CYP3A4. In the case of the methanolic extract, the observed inhibitory effect was significantly stronger than that of artemisinin. This was also confirmed for the extract in tea form, except that no statistically significant difference in the inhibition CYP3A4 was observed in comparison with artemisinin at the highest tested dose of 600 µM. Although these studies were performed on human liver microsomes (HLM) and not on single isolated enzymes as in this case, positive agreement with the results of this study can be confirmed. In addition, further research should be directed toward the study of inhibition kinetics and the determination of kinetic parameters for individually studied enzymes, as well as the effect of AAE in in vivo studies.In this study, a decrease in CYP2B6 enzyme activity was observed by incubation with AAE. Enzyme activity decreased by 88.5% and 60.3%, respectively. These results are consistent with the only available research on the effect of AAE on the CYP2B6 enzyme conducted by Desrosiers et al. . The effThe aforementioned could be explained by the recent results presented by Fu et al. . They inThe most frequently observed type of inhibition of CYP enzymes is direct inhibition . HoweverThese results are particularly important in the context of the last test performed in this study, which confirms the irreversible nature of this inhibition. Enzyme dialysis after incubation failed to restore the activity of CYP2B6 and CYP3A4 enzymes. Addition of the oxidant PCF also did not result in a return of enzyme activity, confirming the view that this case is not a reversible or pseudo-irreversible inhibition. Irreversible inhibitors are much more dangerous in clinically relevant interactions because irreversible damage to the active site occurs in the form of covalent bonds. Such inhibition cannot be stopped by the addition of excess substrate. Therefore, the results of this study regarding the irreversibility of CYP2B6 and CYP3A4 inhibition have strong implications for further in vivo research.Artemisia annua L. is widely used in traditional medicine, all health care professionals and stakeholders should be aware of this inhibition and the potential clinical implications. Further in vivo studies are needed to investigate the interactions of AAE with CYP2B6 and CYP3A4 enzyme substrates.AAE causes inhibition of the CYP2B6 and CYP3A4 enzymes in vitro. Inhibition was most pronounced in metabolism-dependent inhibition assays, followed by direct inhibition assays. No statistically significant enzyme inhibition was observed in time-dependent inhibition assays. In heme-binding assays, AAE was observed to cause a significant decrease in heme concentration, excluding the effect of reactive oxygen species on this decrease. The heme destruction is the result of binding of reactive AAE metabolites to heme, i.e., irreversible inhibition. Irreversible inhibition was confirmed by additional assays, whereas reversible or pseudo-irreversible inhibition could not be confirmed. Since


Pyrococcus horikoschii endoglucanase (PhEG) to tolerate 11 superbase ILs were explored.Ionic liquids (ILs) that dissolve biomass are harmful to the enzymes that degrade lignocellulose. Enzyme hyperthermostability promotes a tolerance to ILs. Therefore, the limits of hyperthemophilic Km increased greatly in the presence ILs, indicating significant competitive inhibition. Increased hydrophobicity of the IL cation or anion was associated with the strongest enzyme inhibition and activation. Surprisingly, PhEG activity was increased 2.0–2.5-fold by several ILs in 4% substrate. Cations exerted the main role in competitive inhibition of the enzyme as revealed by their greater binding energy to the active site.PhEG was found to be most tolerant to 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) in soluble 1% carboxymethylcellulose (CMC) and insoluble 1% Avicel substrates. At 35% concentration, this IL caused an increase in enzyme activity (up to 1.5-fold) with CMC. Several ILs were more enzyme inhibiting with insoluble Avicel than with soluble CMC. These results reveal new ways to design a beneficial combination of ILs and enzymes for the hydrolysis of lignocellulose, and the strong potential of PhEG in industrial, high substrate concentrations in aqueous IL solutions.The online version contains supplementary material available at 10.1007/s10529-022-03268-5. Endo-1,4-β-glucanase acts by randomly hydrolysing cellulose chains  are salts that exist in liquid form below 100 °C and have the capacity to dissolve lignocellulose for improved hydrolysis by enzymes. They are potentially low polluting and display high thermal and chemical stability, high polarity and very low toxicity  (family GH5) was expressed from Escherichia coli and partly purified, as described in Hämäläinen et al.  with organic acids, such as acetate, propionate and guaiacolate  and crystalline cellulose  were measured with the 3,5-dinitrosalicylic acid (DNS) method Miller , as descKinetic parameters were determined in standard assay conditions at 70 °C (pH 6) with and without 5% (v/v) IL. Initial velocities were determined using 4, 6, 10 and 20 mg/ml CMC at different incubation times . Initial velocity was determined from the slope of the graph . All experiments were performed three times with triplicates. Kinetic values were calculated by the hyperbolic regression analysis function (without weighting) in the Hyper 32 program.The IL cations and anions were docked by SwissDock  have been studied earlier, our study revealed new behaviour associated with a hyperthermostable endoglucanase in a range of ILs.2Et showed activity at 35% concentration, although the latter two reduced the activity. At 15% concentration, [mDBN]Me2PO4 also showed clear elevation of PhEG activity. Altogether, [DBNH]OAc, [DBNH]CO2Et and [mDBN]Me2PO4 were well tolerated by PhEG at 15% IL concentration, but activity was rapidly lost at 25% concentration. No consistent trend of inhibition was observed at 25% IL concentration when the cation or the anion were varied , and was less inhibited or even stimulated by the less hydrophobic cations . A similar finding was observed when TfXYN10A was assayed with superbase ILs  superbase ILs. In line with our results, the authors found that the greatest MCC hydrolysis yield was obtained with [EMIM]AOc, followed by [TMGH]AOc, and that the activity was strongly inhibited by [DBNH]OAc and [DBNH]CO2Et.The activity of PhEG was studied in 5–35% acetate-propionate ILs Fig. , and in m et al.  studied 2Et and [DBNH]OAc . Propionate and guaiacolate showed greater activation than acetate when they were conjugated to the less hydrophobic cation [TMGH]+ , or those that do not dissolve cellulose (i.e. guaiacolate ILs)  and insoluble microcrystalline cellulose (Avicel), we tested how ILs affect enzyme activity with these substrates. Comparison of the two substrates revealed two distinct activity patterns. In [DBNH]COtes Fig. . These Ites Fig. , 2014, wtes Fig.  found thIn this study, [EMIM]OAc permitted full activity (even slightly enhanced activity) of PhEG in both substrates at 15% concentration at 70 °C Fig.  observedThe tolerance of PhEG to 35% [EMIM]OAc in hydrolysing crystalline cellulose may have practical importance. It has been shown that relatively low [EMIM]OAc concentrations (25–50% w/v) in water may be effective in pretreating biomass (Fu and Mazza Km (Michaelis constant) and Vmax (maximum reaction velocity) values of the enzymatic reaction were determined in the presence of 5% ILs . This suggests that Vmax reflects the ability of the enzyme to be stimulated when competitive inhibition is relieved. Similarly, enzyme activity in 25% ILs correlates with the relative catalytic efficiency (relative Vmax/KM) in the presence of ILs , which indicates that the theoretical Km and Vmax values in the presence of ILs successfully capture the effects of ILs on enzyme activity. An increase in Km tends to lead to an increase in Vmax, which was clearly seen in the effect of ILs on PhEG activity .There was an approximate linear correlation between the increase in Km and Vmax values or kcat  has also been observed for a hyperthermostable β-glucosidase  was associated with a more compact protein structure . They suggested that this compact structure hinder the approaching of the substrate to the active site, but also enhances the catalytic rate. A simulation study of GH11 xylanase in an aqueous [EMIM]OAc solution suggested that the strong binding of cations in the enzyme active site may potentially reduce or enhance activity by inducing new geometries or electronic structures that affect the transition states  , were not affected or decreased in the presence of this IL , and retained full activity (107%) in [TMGH]n-PrCOO , which suggests a role for hydrophobic interactions in the IL interaction with the active site and in activation of the enzyme. Furthermore, PhEG showed lowered activity in the presence of 15% [Chol]OAc  attached to the cation . The IL cations are trapped at the active site canyon of the enzyme in the same areas where the substrate binds. The binding areas for the [EMIM]+ cation and the cellotetraose substrate are shown in Fig. + cation in a pocket above the catalytic residues of PhEG are shown in Supplementary Fig. 10. The positive charge of [EMIM]+ is located close to the catalytic glutamate side chains of PhEG, as was reported earlier for this enzyme  indicates that as expected the anion with a larger hydrophobic group is better able to fill the PhEG active site canyon , as has also been shown for GH10 xylanase TfXYN10A  are likely to bind to the same active site positions as the sugar ring structures of the substrate . Our docking results indicate that binding of several IL molecules simultaneously onto the active site contributes to the overall effect on enzyme activity, and that there are significant differences in the binding profile between the various IL molecules and between the different enzymes.The ring structures of inhibiting cations Below is the link to the electronic supplementary material.