Disproportionate declines of formerly abundant species underlie insect loss.

Studies have reported widespread declines in terrestrial insect abundances in recent years1-4, but trends in other biodiversity metrics are less clear-cut5-7. Here we examined long-term trends in 923 terrestrial insect assemblages monitored in 106 studies, and found concomitant declines in abundance and species richness. For studies that were resolved to species level (551 sites in 57 studies), we observed a decline in the number of initially abundant species through time, but not in the number of very rare species. At the population level, we found that species that were most abundant at the start of the time series showed the strongest average declines (corrected for regression-to-the-mean effects). Rarer species were, on average, also declining, but these were offset by increases of other species. Our results suggest that the observed decreases in total insect abundance2 can mostly be explained by widespread declines of formerly abundant species. This counters the common narrative that biodiversity loss is mostly characterized by declines of rare species8,9. Although our results suggest that fundamental changes are occurring in insect assemblages, it is important to recognize that they represent only trends from those locations for which sufficient long-term data are available. Nevertheless, given the importance of abundant species in ecosystems10, their general declines are likely to have broad repercussions for food webs and ecosystem functioning.

The recovery of European freshwater biodiversity has come to a halt.

Owing to a long history of anthropogenic pressures, freshwater ecosystems are among the most vulnerable to biodiversity loss1. Mitigation measures, including wastewater treatment and hydromorphological restoration, have aimed to improve environmental quality and foster the recovery of freshwater biodiversity2. Here, using 1,816 time series of freshwater invertebrate communities collected across 22 European countries between 1968 and 2020, we quantified temporal trends in taxonomic and functional diversity and their responses to environmental pressures and gradients. We observed overall increases in taxon richness (0.73% per year), functional richness (2.4% per year) and abundance (1.17% per year). However, these increases primarily occurred before the 2010s, and have since plateaued. Freshwater communities downstream of dams, urban areas and cropland were less likely to experience recovery. Communities at sites with faster rates of warming had fewer gains in taxon richness, functional richness and abundance. Although biodiversity gains in the 1990s and 2000s probably reflect the effectiveness of water-quality improvements and restoration projects, the decelerating trajectory in the 2010s suggests that the current measures offer diminishing returns. Given new and persistent pressures on freshwater ecosystems, including emerging pollutants, climate change and the spread of invasive species, we call for additional mitigation to revive the recovery of freshwater biodiversity.

Actin and CDC-42 contribute to nuclear migration through constricted spaces in C. elegans.

Successful nuclear migration through constricted spaces between cells or in the extracellular matrix relies on the ability of the nucleus to deform. Little is known about how this takes place in vivo. We have studied confined nuclear migration in Caenorhabditis elegans larval P cells, which is mediated by the LINC complex to pull nuclei towards the minus ends of microtubules. Null mutations of the LINC component unc-84 lead to a temperature-dependent phenotype, suggesting a parallel pathway for P-cell nuclear migration. A forward genetic screen for enhancers of unc-84 identified cgef-1 (CDC-42 guanine nucleotide exchange factor). Knockdown of CDC-42 in the absence of the LINC complex led to a P-cell nuclear migration defect. Expression of constitutively active CDC-42 partially rescued nuclear migration in cgef-1; unc-84 double mutants, suggesting that CDC-42 functions downstream of CGEF-1. The Arp2/3 complex and non-muscle myosin II (NMY-2) were also found to function parallel to the LINC pathway. In our model, CGEF-1 activates CDC-42, which induces actin polymerization through the Arp2/3 complex to deform the nucleus during nuclear migration, and NMY-2 helps to push the nucleus through confined spaces.

Altered integration of excitatory inputs onto the basal dendrites of layer 5 pyramidal neurons in a mouse model of Fragile X syndrome.

Layer 5 (L5) pyramidal neurons receive predictive and sensory inputs in a compartmentalized manner at their apical and basal dendrites, respectively. To uncover how integration of sensory inputs is affected in autism spectrum disorders (ASD), we used two-photon glutamate uncaging to activate spines in the basal dendrites of L5 pyramidal neurons from a mouse model of Fragile X syndrome (FXS), the most common genetic cause of ASD. While subthreshold excitatory inputs integrate linearly in wild-type animals, surprisingly those with FXS summate sublinearly, contradicting what would be expected of sensory hypersensitivity classically associated with ASD. We next investigated the mechanism underlying this sublinearity by performing knockdown of the regulatory ß4 subunit of BK channels, which rescued the synaptic integration, a result that was corroborated with numerical simulations. Taken together, these findings suggest that there is a differential impairment in the integration of feedforward sensory and feedback predictive inputs in L5 pyramidal neurons in FXS and potentially other forms of ASD, as a result of specifically localized subcellular channelopathies. These results challenge the traditional view that FXS and other ASD are characterized by sensory hypersensitivity, proposing instead a hyposensitivity of sensory inputs and hypersensitivity of predictive inputs onto cortical neurons.

A PDX1 cistrome and single-cell transcriptome resource of the developing pancreas.

Pancreatic and duodenal homeobox 1 (PDX1) is crucial for pancreas organogenesis, yet the dynamic changes in PDX1 binding in human or mouse developing pancreas have not been examined. To address this knowledge gap, we performed PDX1 ChIP-seq and single-cell RNA-seq using fetal human pancreata. We integrated our datasets with published datasets and revealed the dynamics of PDX1 binding and potential cell lineage-specific PDX1-bound genes in the pancreas from fetal to adult stages. We identified a core set of developmentally conserved PDX1-bound genes that reveal the broad multifaceted role of PDX1 in pancreas development. Despite the well-known dramatic changes in PDX1 function and expression, we found that PDX1-bound genes are largely conserved from embryonic to adult stages. This points towards a dual role of PDX1 in regulating the expression of its targets at different ages, dependent on other functionally congruent or directly interacting partners. We also showed that PDX1 binding is largely conserved in mouse pancreas. Together, our study reveals PDX1 targets in the developing pancreas in vivo and provides an essential resource for future studies on pancreas development.

Aging and sperm signals alter DNA break formation and repair in the C. elegans germline.

Female reproductive aging is associated with decreased oocyte quality and fertility. The nematode Caenorhabditis elegans is a powerful system for understanding the biology of aging and exhibits age-related reproductive defects that are analogous to those observed in many mammals, including dysregulation of DNA repair. C. elegans germline function is influenced simultaneously by both reproductive aging and signals triggered by limited supplies of sperm, which are depleted over chronological time. To delineate the causes of DNA repair defects in aged C. elegans germlines, we assessed both DNA double strand break (DSB) induction and repair during meiotic prophase I progression in aged germlines which were depleted of self-sperm, mated, or never exposed to sperm. We find that germline DSB induction is dramatically reduced only in hermaphrodites which have exhausted their endogenous sperm, suggesting that a signal due specifically to sperm depletion downregulates DSB formation. We also find that DSB repair is delayed in aged germlines regardless of whether hermaphrodites had either a reduction in sperm supply or an inability to endogenously produce sperm. These results demonstrate that in contrast to DSB induction, DSB repair defects are a feature of C. elegans reproductive aging independent of sperm presence. Finally, we demonstrate that the E2 ubiquitin-conjugating enzyme variant UEV-2 is required for efficient DSB repair specifically in young germlines, implicating UEV-2 in the regulation of DNA repair during reproductive aging. In summary, our study demonstrates that DNA repair defects are a feature of C. elegans reproductive aging and uncovers parallel mechanisms regulating efficient DSB formation in the germline.

The Neural Basis for Biased Behavioral Responses Evoked by Galvanic Vestibular Stimulation in Primates.

Noninvasive electrical stimulation of the vestibular system in humans has become an increasingly popular tool with a broad range of research and clinical applications. However, common assumptions regarding the neural mechanisms that underlie the activation of central vestibular pathways through such stimulation, known as galvanic vestibular stimulation (GVS), have not been directly tested. Here, we show that GVS is encoded by VIIIth nerve vestibular afferents with nonlinear dynamics that differ markedly from those predicted by current models. GVS produced asymmetric activation of both semicircular canal and otolith afferents to the onset versus offset and cathode versus anode of applied current, that in turn produced asymmetric eye movement responses in three awake-behaving male monkeys. Additionally, using computational methods, we demonstrate that the experimentally observed nonlinear neural response dynamics lead to an unexpected directional bias in the net population response when the information from both vestibular nerves is centrally integrated. Together our findings reveal the neural basis by which GVS activates the vestibular system, establish that neural response dynamics differ markedly from current predictions, and advance our mechanistic understanding of how asymmetric activation of the peripheral vestibular system alters vestibular function. We suggest that such nonlinear encoding is a general feature of neural processing that will be common across different noninvasive electrical stimulation approaches.SIGNIFICANCE STATEMENT Here, we show that the application of noninvasive electrical currents to the vestibular system (GVS) induces more complex responses than commonly assumed. We recorded vestibular afferent activity in macaque monkeys exposed to GVS using a setup analogous to human studies. GVS evoked notable asymmetries in irregular afferent responses to cathodal versus anodal currents. We developed a nonlinear model explaining these GVS-evoked afferent responses. Our model predicts that GVS induces directional biases in centrally integrated head motion signals and establishes electrical stimuli that recreate physiologically plausible sensations of motion. Altogether, our findings provide new insights into how GVS activates the vestibular system, which will be vital to advancing new clinical and biomedical applications.

Phosphorylation Mechanism Switching in Histidine Kinases Is a Tool for Fast Protein Evolution: Insights From AlphaFold Models.

Histidine kinases (HKs) are a central part of bacterial environmental-sensing two-component systems. They provide their hosts with the ability to respond to a wide range of physical and chemical signals. HKs are multidomain proteins consisting of at least a sensor domain, dimerization and phosphorylation domain (DHp), and a catalytic domain. They work as homodimers and the existence of two different autophosphorylation mechanisms (cis and trans) has been proposed as relevant for pathway specificity. Although several HKs have been intensively studied, a precise sequence-to-structure explanation of why and how either cis or trans phosphorylation occurs is still unavailable nor is there any evolutionary analysis on the subject. In this work, we show that AlphaFold can accurately determine whether an HK dimerizes in a cis or trans structure. By modeling multiple HKs we show that both cis- and trans-acting HKs are common in nature and the switch between mechanisms has happened multiple times in the evolutionary history of the family. We then use AlphaFold modeling to explore the molecular determinants of the phosphorylation mechanism. We conclude that it is the difference in lengths of the helices surrounding the DHp loop that determines the mechanism. We also show that very small changes in these helices can cause a mechanism switch. Despite this, previous evidence shows that for a particular HK the phosphorylation mechanism is conserved. This suggests that the phosphorylation mechanism participates in system specificity and mechanism switching provides these systems with a way to diverge.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Oral minoxidil for late alopecia in cancer survivors.

PURPOSE: Late alopecia, defined as incomplete hair regrowth > 6 months following cytotoxic chemotherapy or > 6 months from initiation of endocrine therapy, negatively impacts quality of life and may affect dose intensity of adjuvant therapy. This study investigates the effect of oral minoxidil in women with chemotherapy and/or endocrine therapy-induced late alopecia. METHODS: The rate of clinical response was assessed by standardized photography and quantitated with trichoscopy. RESULTS: Two hundred and sixteen patients (mean age 57.8 ± 13.7) were included. The most common cancer diagnosis was breast, in 170 patients (79.1%). Alopecia developed after chemotherapy in 31 (14.4%) patients, endocrine monotherapy in 65 (30.1%) patients, and chemotherapy followed by endocrine therapy in 120 (55.6%) patients. In 119 patients, standardized photography assessments were used to determine clinical change in alopecia after a median of 105 (IQR = 70) days on oral minoxidil and revealed improvement in 88 (74%) patients. Forty-two patients received quantitative trichoscopic assessments at baseline and at follow-up after a median of 91 (IQR = 126) days on oral minoxidil. Patients had clinically and statistically significant increases in frontal hair shaft density (from 124.2 hairs/cm2 at initial to 153.2 hairs/cm2 at follow-up assessment, p = 0.008) and occipital shaft density (from 100.3 hairs/cm2 at initial to 123.5 hairs/cm2 at follow-up assessment. p = 0.004). No patients discontinued oral minoxidil due to adverse events. CONCLUSIONS: Overall, oral minoxidil was well tolerated by patients and may benefit both frontal and occipital late alopecia in cancer survivors treated with cytotoxic and/or endocrine therapy by increasing hair shaft and follicle density.

The Potential of Cannabichromene (CBC) as a Therapeutic Agent.

There is a growing interest in the use of medicinal plants to treat a variety of diseases, and one of the most commonly used medicinal plants globally is Cannabis sativa The two most abundant cannabinoids (Δ9-tetrahydrocannabinol and cannabidiol) have been governmentally approved to treat selected medical conditions; however, the plant produces over 100 cannabinoids, including cannabichromene (CBC). While the cannabinoids share a common precursor molecule, cannabigerol, they are structurally and pharmacologically unique. These differences may engender differing therapeutic potentials. In this review, we will examine what is currently known about CBC with regards to pharmacodynamics, pharmacokinetics, and receptor profile. We will also discuss the therapeutic areas that have been examined for this cannabinoid, notably antinociceptive, antibacterial, and anti-seizure activities. Finally, we will discuss areas where new research is needed and potential novel medicinal applications for CBC. Significance Statement Cannabichromene (CBC) has been suggested to have disparate therapeutic benefits such as anti-inflammatory, anticonvulsant, antibacterial, and antinociceptive effects. Most of the focus on the medical benefits of cannabinoids has been focused on THC and CBD. The preliminary studies on CBC indicate that this phytocannabinoid may have unique therapeutic potential that warrants further investigation. Following easier access to hemp, CBC products are commercially available over-the-counter and are being widely utilized with little or no evidence of their safety or efficacy.

High Cannabigerol Hemp Extract Moderates Colitis and Modulates the Microbiome in an Inflammatory Bowel Disease Model.

Cannabis sativa L. has a long history of medicinal use, particularly for gastrointestinal diseases. Patients with inflammatory bowel disease (IBD) report using cannabis to manage their symptoms, despite little data to support the use of cannabis or cannabis products to treat the disease. In this study, we use the well-described dextran sodium sulfate (DSS) model of colitis in mice to assess the impact of commercially available, noneuphorigenic, high cannabigerol (CBG) hemp extract (20 mg/mL cannabigerol, 20.7 mg/mL cannabidiol, 1 mg/mL cannabichromene) on IBD activity and the colonic microbiome. Mice were given 2% DSS in drinking water for 5 days, followed by 2 days of regular drinking water. Over the 7 days, mice were dosed daily with either high CBG hemp extract or matched vehicle control. Daily treatment with high CBG hemp extract dramatically reduces the severity of disease at the histological and organismal levels as measured by decreased disease activity index, increased colon length, and decreases in percent colon tissue damage. 16S rRNA gene sequencing of the fecal microbiota reveals high CBG hemp extract treatment results in alterations in the microbiota that may be beneficial for colitis. Finally, using metabolomic analysis of fecal pellets, we find that mice treated with high CBG hemp extract have a normalization of several metabolic pathways, including those involved in inflammation. Taken together, these data suggest that high CBG hemp extracts may offer a novel treatment option for patients. SIGNIFICANCE STATEMENT: Using the dextran sodium sulfate model of colitis, the authors show that treatment with high cannabigerol hemp extract reduces the severity of symptoms associated with colitis. Additionally, they show that treatment modulates both the fecal microbiota and metabolome with potential functional significance.

Extreme γ' fibrinogen levels in COVID-19 patients.

COVID-19 disease progression can be accompanied by a "cytokine storm" that leads to secondary sequelae such as acute respiratory distress syndrome. Several inflammatory cytokines have been associated with COVID-19 disease progression, but have high daily intra-individual variability. In contrast, we have shown that the inflammatory biomarker γ' fibrinogen (GPF) has a 6-fold lower coefficient of variability compared to other inflammatory markers such as hs-CRP. The aims of the study were to measure GPF in serial blood samples from COVID-19 patients at a tertiary care medical center in order to investigate its association with clinical measures of disease progression. COVID-19 patients were retrospectively enrolled between 3/16/2020 and 8/1/2020. GPF was measured using a commercial ELISA. We found that COVID-19 patients can develop extraordinarily high levels of GPF. Our results showed that ten out of the eighteen patients with COVID-19 had the highest levels of GPF ever recorded. The previous highest GPF level of 80.3 mg/dL was found in a study of 10,601 participants in the ARIC study. GPF levels were significantly associated with the need for ECMO and mortality. These findings have potential implications regarding prophylactic anticoagulation of COVID-19 patients.

Cytochrome c levels affect the TOR pathway to regulate growth and metabolism under energy-deficient conditions.

Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.

An intuitive approach for measuring the resilience of biological communities to environmental change.

Research Highlight: Saether, B. E., Engen, S., & Solbu, E. B. (2023a). Assessing the sensitivity and resistance of communities in a changing environment. Journal of Animal Ecology. https://doi.org/10.1111/1365-2656.14003. In the face of global change, conservation strategies can be informed by understanding which biological communities are most at risk. Metrics that reflect the 'resilience' of communities to change could have great utility, but there is still no consensus on the most useful way to measure it. Saether et al. introduce an intuitive approach to thinking about and measuring resilience based on how variation in the total number of individuals within a community affects the number of species. By using dynamic species abundance distribution models, they also quantify the different sources of population-level variation that contribute to community resilience. Evenness emerges as an important predictor of resilience, with more even communities predicted to be more sensitive to abundance loss. An attractive feature of their approach is the ability to estimate the key parameters using commonly used generalized linear mixed effects models, which they illustrate with a case study on forest bird communities. The approach is ripe for comparison across different systems to explore how these proposed metrics complement existing biodiversity metrics and how they help understand the risk of communities from environmental change.

The MAGIC trial: a pragmatic, multicentre, parallel, noninferiority, randomised trial of melatonin versus midazolam in the premedication of anxious children attending for elective surgery under general anaesthesia.

BACKGROUND: Child anxiety before general anaesthesia and surgery is common. Midazolam is a commonly used premedication to address this. Melatonin is an alternative anxiolytic, however trials evaluating its efficacy in children have delivered conflicting results. METHODS: This multicentre, double-blind randomised trial was performed in 20 UK NHS Trusts. A sample size of 624 was required to declare noninferiority of melatonin. Anxious children, awaiting day case elective surgery under general anaesthesia, were randomly assigned 1:1 to midazolam or melatonin premedication (0.5 mg kg-1, maximum 20 mg) 30 min before transfer to the operating room. The primary outcome was the modified Yale Preoperative Anxiety Scale-Short Form (mYPAS-SF). Secondary outcomes included safety. Results are presented as n (%) and adjusted mean differences with 95% confidence intervals. RESULTS: The trial was stopped prematurely (n=110; 55 per group) because of recruitment futility. Participants had a median age of 7 (6-10) yr, and 57 (52%) were female. Intention-to-treat and per-protocol modified Yale Preoperative Anxiety Scale-Short Form analyses showed adjusted mean differences of 13.1 (3.7-22.4) and 12.9 (3.1-22.6), respectively, in favour of midazolam. The upper 95% confidence interval limits exceeded the predefined margin of 4.3 in both cases, whereas the lower 95% confidence interval excluded zero, indicating that melatonin was inferior to midazolam, with a difference considered to be clinically relevant. No serious adverse events were seen in either arm. CONCLUSION: Melatonin was less effective than midazolam at reducing preoperative anxiety in children, although the early termination of the trial increases the likelihood of bias. CLINICAL TRIAL REGISTRATION: ISRCTN registry: ISRCTN18296119.

Safety and oncological effectiveness after desensitization in patients with previous hypersensitivity reactions to chemotherapy.

INTRODUCTION: Taxanes and platinum are first-line treatments in gynecological tumors with high rates of hypersensitivity reactions (HSRs), leading to discontinuation of treatment. Desensitization involves induction of temporary tolerance to previously sensitized medications. The aims of this study are to describe HSRs to paclitaxel and carboplatin and evaluate the safety and effectiveness of desensitization protocols in gynecological cancer patients. METHODS: Original, retrospective, descriptive, analytical study, approved by Bioethics and Research Committee, included >18-year-old patients with gynecological tumors experiencing HSRs to first-line chemotherapy. Patients underwent 3-bag-12-step desensitization. RESULTS: 174 desensitization (95 paclitaxel, 79 carboplatin) in 33 female patients, mean age 45.5 years (18-71y). Cancer diagnosis: breast 8 (24.2%), ovarian 14 (42.2%), endometrial 2 (6.1%) and cervix 9 (27.2%). HSR occurred in paclitaxel during cycles 1-2 and in carboplatin after 6 cycles. The most frequently seen HSR symptom was cardiovascular with paclitaxel (94.7%), and cutaneous (93.3%) with carboplatin. Three-bags 12-steps desensitization protocol (initial dilution 1:100) in 5.67hrs. All patients reached total dose desensitization: 82% with no reaction, 12% mild, 6% moderate and 0% severe reaction. Mean disease-free interval and progression-free interval in months (m): breast cancer 29 m and 14 m, ovarian 22 m and 9 m, endometrial 40 m and cervical cancer: 67.5 m and 27 m. Twenty-five patients (73.5%) are still alive. CONCLUSION: HSRs to paclitaxel manifest in the first 1-2 cycles and to carboplatin after 6 cycles. Symptoms include cardiovascular, atypical neuromuscular and urticaria. Changing treatment lines impacts prognosis. Our study revealed that ovarian cancer patients undergoing desensitization protocols achieved longer progression-free intervals. All patients successfully reached total dose desensitization. This study provides evidence of the effectiveness and safety of desensitization and promising perspective for continuing first-line treatment with HSRs.

Transdermal application of diacerin loaded-terpene enriched invasomes: an approach to augment anti-edema and nociception inhibition activity.

This study aimed to formulate diacerein loaded terpene-enriched invasomes (DCN-TINV) to fulfill a fruitful management of osteoarthritis. A 23 factorial design was adopted, including A: cholesterol concentration (%w/v), B: ethanol volume (mL) and C: phosphatidylcholine: drug ratio as the studied factors. Invasomes were constructed using the thin film hydration technique. Herein, percent entrapment efficiency (EE%), particle size (PS), poly-dispersity index (PDI) and zeta potential (ZP) were statistically analyzed using Design-Expert® software to select the optimum formula. The selected criteria for detecting the optimum formula were restricting PS (<350 nm), dismissing PDI, magnifying ZP (as absolute value) and EE%. The selected formula was further scrutinized through multiple in-vitro studies, including Fourier-transform infrared spectroscopy, differential scanning calorimetry, pH measurement, stability study, release profile and transmission electron microscopy. Furthermore, the ex-vivo performance was evaluated through ex-vivo skin permeation and deposition. Finally, it was subjected to an array of in-vivo tests, namely Draize test, histopathology, In-vivo skin penetration, edema size, and nociception inhibition measurements. The optimum formula with desirability (0.913) demonstrated EE% (89.21% ± 2.12%), PS (319.75 ± 10.11 nm), ZP (-55 ± 3.96 mV) and a prolonged release profile. Intriguingly, revamped skin permeation (1143 ± 32.11 µg/cm2), nociception inhibition (77%) and In-vivo skin penetration (144 µm) compared to DCN suspension (285 ± 21.25 µg/cm2, 26% and 48 µm, respectively) were displayed. The optimum DCN-TINV exhibited plausible safety and stability profiles consolidated with auspicious efficacy for better management of osteoarthritis.

Cannabinoid Tolerance in S426A/S430A x ß-Arrestin 2 Knockout Double-Mutant Mice.

Tolerance to compounds that target G protein-coupled receptors (GPCRs), such as the cannabinoid type-1 receptor (CB1R), is in part facilitated by receptor desensitization. Processes that mediate CB1R desensitization include phosphorylation of CB1R residues S426 and S430 by a GPCR kinase and subsequent recruitment of the ß-arrestin2 scaffolding protein. Tolerance to cannabinoid drugs is reduced in S426A/S430A mutant mice and ß-arrestin2 knockout (KO) mice according to previous work in vivo. However, the presence of additional phosphorylatable residues on the CB1R C-terminus made it unclear as to whether recruitment to S426 and S430 accounted for all desensitization and tolerance by ß-arrestin2. Therefore, we assessed acute response and tolerance to the cannabinoids delta-9-tetrahydrocannabinol (Δ9-THC) and CP55,940 in S426A/S430A x ß-arrestin2 KO double-mutant mice. We observed both delayed tolerance and increased sensitivity to the antinociceptive and hypothermic effects of CP55,940 in male S426A/S430A single- and double-mutant mice compared with wild-type littermates, but not with Δ9-THC. Female S426A/S430A single- and double-mutant mice were more sensitive to acute antinociception (CP55,940 and Δ9-THC) and hypothermia (CP55,940 only) exclusively after chronic dosing and did not differ in the development of tolerance. These results indicate that phosphorylation of S426 and S430 are likely responsible for ß-arrestin2-mediated desensitization as double-mutant mice did not differ from the S426A/S430A single-mutant model in respect to cannabinoid tolerance and sensitivity. We also found antinociceptive and hypothermic effects from cannabinoid treatment demonstrated by sex-, agonist-, and duration-dependent features. SIGNIFICANCE STATEMENT: A better understanding of the molecular mechanisms involved in tolerance will improve the therapeutic potential of cannabinoid drugs. This study determined that further deletion of ß-arrestin2 does not enhance the delay in cannabinoid tolerance observed in CB1R S426A/S430A mutant mice.

Convergent Community Assembly among Globally Separated Acidic Cave Biofilms.

Acidophilic bacteria and archaea inhabit extreme geochemical "islands" that can tell us when and how geographic barriers affect the biogeography of microorganisms. Here, we describe microbial communities from extremely acidic (pH 0 to 1) biofilms, known as snottites, from hydrogen sulfide-rich caves. Given the extreme acidity and subsurface location of these biofilms, and in light of earlier work showing strong geographic patterns among snottite Acidithiobacillus populations, we investigated their structure and diversity in order to understand how geography might impact community assembly. We used 16S rRNA gene cloning and fluorescence in situ hybridization (FISH) to investigate 26 snottite samples from four sulfidic caves in Italy and Mexico. All samples had very low biodiversity and were dominated by sulfur-oxidizing bacteria in the genus Acidithiobacillus. Ferroplasma and other archaea in the Thermoplasmatales ranged from 0 to 50% of total cells, and relatives of the bacterial genera Acidimicrobium and Ferrimicrobium were up to 15% of total cells. Rare phylotypes included Sulfobacillus spp. and members of the phyla "Candidatus Dependentiae" and "Candidatus Saccharibacteria" (formerly TM6 and TM7). Although the same genera of acidophiles occurred in snottites on separate continents, most members of those genera represent substantially divergent populations, with 16S rRNA genes that are only 95 to 98% similar. Our findings are consistent with a model of community assembly where sulfidic caves are stochastically colonized by microorganisms from local sources, which are strongly filtered through environmental selection for extreme acid tolerance, and these different colonization histories are maintained by dispersal restrictions within and among caves. IMPORTANCE Microorganisms that are adapted to extremely acidic conditions, known as extreme acidophiles, are catalysts for rock weathering, metal cycling, and mineral formation in naturally acidic environments. They are also important drivers of large-scale industrial processes such as biomining and contaminant remediation. Understanding the factors that govern their ecology and distribution can help us better predict and utilize their activities in natural and engineered systems. However, extremely acidic habitats are unusual in that they are almost always isolated within circumneutral landscapes. So where did their acid-adapted inhabitants come from, and how do new colonists arrive and become established? In this study, we took advantage of a unique natural experiment in Earth's subsurface to show how isolation may have played a role in the colonization history, community assembly, and diversity of highly acidic microbial biofilms.