From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Sustainability in the IVF laboratory: recommendations of an expert panel.

The healthcare industry is a major contributor to greenhouse gas emissions. Assisted reproductive technology is part of the larger healthcare sector, with its own heavy carbon footprint. The social, economic and environmental costs of this collective carbon footprint are becoming clearer, as is the impact on human reproductive health. Alpha Scientists in Reproductive Medicine and the International IVF Initiative collaborated to seek and formulate practical recommendations for sustainability in IVF laboratories. An international panel of experts, enthusiasts and professionals in reproductive medicine, environmental science, architecture, biorepository and law convened to discuss the topics of importance to sustainability. Recommendations were issued on how to build a culture of sustainability in the workplace, implement green design and building, use life cycle analysis to determine the environmental impact, manage cryostorage more sustainably, and understand and manage laboratory waste with prevention as a primary goal. The panel explored whether the industry supporting IVF is sustainable. An example is provided to illustrate the application of green principles to an IVF laboratory through a certification programme. The UK legislative landscape surrounding sustainability is also discussed and a few recommendations on 'Green Conferencing' are offered.

The Internet of Things in assisted reproduction.

The Internet of Things (IoT) is a network connecting physical objects with sensors, software and internet connectivity for data exchange. Integrating the IoT with medical devices shows promise in healthcare, particularly in IVF laboratories. By leveraging telecommunications, cybersecurity, data management and intelligent systems, the IoT can enable a data-driven laboratory with automation, improved conditions, personalized treatment and efficient workflows. The integration of 5G technology ensures fast and reliable connectivity for real-time data transmission, while blockchain technology secures patient data. Fog computing reduces latency and enables real-time analytics. Microelectromechanical systems enable wearable IoT and miniaturized monitoring devices for tracking IVF processes. However, challenges such as security risks and network issues must be addressed through cybersecurity measures and networking advancements. Clinical embryologists should maintain their expertise and knowledge for safety and oversight, even with IoT in the IVF laboratory.

Cryostorage management of reproductive cells and tissues in ART: status, needs, opportunities and potential new challenges.

Among the wide range of procedures performed by clinical embryologists, the cryopreservation of reproductive cells and tissues represents a fundamental task in the daily routine. Indeed, cryopreservation procedures can be considered a subspecialty of medically assisted reproductive technology (ART), having the same relevance as sperm injection or embryo biopsy for preimplantation genetic testing. However, although a great deal of care has been devoted to optimizing cryopreservation protocols, the same energy has only recently been spent on developing and implementing strategies for the safe and reliable storage and transport of reproductive specimens. Herein, we have summarized the content of the available guidelines, the risks, the needs and the future perspectives regarding the management of cryopreservation biorepositories used in ART.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Cell viability and cytotoxicity of inkjet-printed flexible organic electrodes on parylene C.

This study reports on the fabrication of biocompatible organic devices by means of inkjet printing with a novel combination of materials. The devices were fabricated on Parylene C (PaC), a biocompatible and flexible polymer substrate. The contact tracks were inkjet-printed using a silver nanoparticle ink, while the active sites were inkjet-printed using a poly (3,4ethylenedioxythiophene)/polystyrene sulfonate (PEDOT:PSS) solution. To insulate the final device, a polyimide ink was used to print a thick film, leaving small open windows upon the active sites. Electrical characterization of the final device revealed conductivities in the order of 103 and 102 S.cm-1 for Ag and PEDOT based inks, respectively. Cell adhesion assays performed with PC-12 cells after 96 h of culture, and B16F10 cells after 24 h of culture, demonstrated that the cells adhered on top of the inks and cell differentiation occurred, which indicates Polyimide and PEDOT:PSS inks are non-toxic to these cells. The results indicate that PaC, along with its surface-treated variants, is a potentially useful material for fabricating cell-based microdevices.

Safety, pharmacokinetics and pharmacodynamics of HTL0009936, a selective muscarinic M1 -acetylcholine receptor agonist: A randomized cross-over trial.

AIMS: HTL0009936 is a selective M1 muscarinic receptor agonist in development for cognitive dysfunction in Alzheimer's disease. Safety, tolerability and pharmacokinetics and exploratory pharmacodynamic effects of HTL0009936 administered by continuous IV infusion at steady state were investigated in elderly subjects with below average cognitive functioning (BACF). METHODS: Part A was a four-treatment open label sequential study in healthy elderly investigating 10-83 mg HTL0009936 (IV) and a 24 mg HTL0009936 single oral dose. Part B was a five-treatment randomized, double-blind, placebo and physostigmine controlled cross-over study with IV HTL0009936 in elderly subjects with BACF. Pharmacodynamic assessments were performed using neurocognitive and electrophysiological tests. RESULTS: Pharmacokinetics of HTL0009936 showed dose-proportional increases in exposure with a mean half-life of 2.4 hours. HTL0009936 was well-tolerated with transient dose-related adverse events (AEs). Small increases in mean systolic blood pressure of 7.12 mmHg (95% CI [3.99-10.24]) and in diastolic of 5.32 mmHg (95% CI [3.18-7.47]) were noted at the highest dose in part B. Overall, there was suggestive, but no definitive, positive or negative pharmacodynamic effects. Statistically significant effects were observed on P300 with HTL0009936 and adaptive tracking with physostigmine. CONCLUSIONS: HTL0009936 showed well-characterized pharmacokinetics and single doses were safe and generally well-tolerated in healthy elderly subjects. Due to physostigmine tolerability issues and subject burden, the study design was changed and some pharmacodynamic assessments (neurocognitive) were performed at suboptimal drug exposures. Therefore no clear conclusions can be made on pharmacodynamic effects of HTL0009936, although an effect on P300 is suggestive of central target engagement.

First-in-man study to investigate safety, pharmacokinetics and exploratory pharmacodynamics of HTL0018318, a novel M1 -receptor partial agonist for the treatment of dementias.

AIMS: HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. METHODS: This randomized, double-blind, placebo-controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1-35 mg HTL0018318. Pharmacodynamic assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. Cerebrospinal fluid concentrations of HTL0018318 and food effects on pharmacokinetics of HTL0018318 were investigated in an open label and partial cross-over design in 14 healthy subjects. RESULTS: Pharmacokinetics of HTL0018318 were well-characterized showing dose proportional increases in exposure from 1-35 mg. Single doses of HTL0018318 were associated with mild dose-related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mmHg in younger adults (95% CI [4.2-16.3], 35-mg dose) and up to 11.9 mmHg in elderly subjects (95% CI [4.9-18.9], 15-mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. CONCLUSION: HTL0018318 showed well-characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for Alzheimer's disease and other dementias.

Chronic diarrhoea in adults: what not to miss.

PURPOSE OF REVIEW: Chronic diarrhoea remains a diagnostic challenge, with numerous causes and few effective symptomatic treatments. This review focuses on new methods for diagnosis of common disorders and alerts readers to rarer causes through a systematic approach to the underlying mechanisms. RECENT FINDINGS: New strategies are emerging to stratify the need for endoscopic investigation. Faecal immunochemical testing, combined with standard blood tests, shows promise in excluding colorectal cancers, adenoma and inflammatory bowel disease, challenging the current use of faecal calprotectin. Serum analysis for markers of bile acid synthesis has been refined, potentially streamlining diagnostic pathways of bile acid malabsorption for those who are unable to access nuclear medicine scans, but the positive predictive value of faecal elastase in low prevalence populations has been questioned. Novel markers such as volatile organic compounds and stool DNA analyses continue to develop. SUMMARY: A systematic approach to investigation of chronic diarrhoea will ensure all relevant causes are considered and minimize the chance of a missed diagnosis. Combination of clinical features with noninvasive testing supports a judicious approach to endoscopic investigations but further innovation will be needed to resolve the diagnostic challenge that diarrhoea poses.

Comparison of 36 assisted reproduction laboratories monitoring environmental conditions and instrument parameters using the same quality-control application.

RESEARCH QUESTION: Assisted reproduction laboratories record instrument performance periodically. No standardized guidelines have been produced for this activity despite mandatory auditing systems in several countries. This study of 36 laboratories in 12 different countries was conducted to assess differences and similarities between quality assurance programmes using an adaptable cloud-based quality-control app for instrument monitoring. DESIGN: A total of 36 deidentified IVF laboratories that subscribed to the same quality-assurance application were studied. Data were evaluated based on instrument types allocated to 10 domains: incubators, gas tanks, warming surfaces, refrigerators and freezers, cryo-storage, environment, water purification, peripheral equipment, checklists and miscellaneous. RESULTS: The incubator domain constituted the greatest proportion of parameters (35%), followed by surface warming instruments at 15%. Most incubator O2 readings were monitored between 4.5 and 5.5%, and between 5.5 and 6.5% for CO2. The altitude of the laboratory was poorly correlated with the CO2 setting. Incubator display and measured values of gases and temperature by built-in sensors vary considerably compared with third-party sensors. A quality-control diligence score or mean average data points was calculated for each laboratory. This score is independent of number of instruments or laboratory size. Higher scores were associated with laboratories in countries with government regulations and mandatory auditing systems. CONCLUSIONS: Major differences exist in instrument monitoring practices among laboratories. Although incubator monitoring is the largest domain, many other sensitive instruments are diligently monitored by most laboratories. International standardization and guidelines are needed.

Identification of a novel allosteric GLP-1R antagonist HTL26119 using structure- based drug design.

A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C3476.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.

Coinfection and Emergence of Rifamycin Resistance during a Recurrent Clostridium difficile Infection.

Clostridium difficile (Peptoclostridium difficile) is a common health care-associated infection with a disproportionately high incidence in elderly patients. Disease symptoms range from mild diarrhea to life-threatening pseudomembranous colitis. Around 20% of patients may suffer recurrent disease, which often requires rehospitalization of patients. C. difficile was isolated from stool samples from a patient with two recurrent C. difficile infections. PCR ribotyping, whole-genome sequencing, and phenotypic assays were used to characterize these isolates. Genotypic and phenotypic screening of C. difficile isolates revealed multiple PCR ribotypes present and the emergence of rifamycin resistance during the infection cycle. Understanding both the clinical and bacterial factors that contribute to the course of recurrent infection could inform strategies to reduce recurrence. (This study has been registered at ClinicalTrials.gov under registration no. NCT01670149.).

Endocannabinoids, through opioids and prostaglandins, contribute to fever induced by key pyrogenic mediators.

This study aims to explore the contribution of endocannabinoids on the cascade of mediators involved in LPS-induced fever and to verify the participation of prostaglandins and endogenous opioids in fever induced by anandamide (AEA). Body temperature (Tc) of male Wistar rats was recorded over 6h, using a thermistor probe. Cerebrospinal fluid concentration of PGE2 and ß-endorphin were measured by ELISA after the administration of AEA. Intracerebroventricular administration of the CB1 receptor antagonist AM251 (5µg, i.c.v.), reduced the fever induced by IL-1ß (3ng, i.c.v.), TNF-α (250ng, i.c.v.), IL-6 (300ng, i.c.v.), corticotrophin release factor (CRH; 2.5µg, i.c.v.) and endothelin (ET)-1 (1pmol, i.c.v.), but not the fever induced by PGE2 (250ng, i.c.v.) or PGF2α (250ng, i.c.v.). Systemic administration of indomethacin (2mgkg(-1), i.p.) or celecoxib (5mgkg(-1), p.o.) reduced the fever induced by AEA (1µg, i.c.v.), while naloxone (1mgkg(-1), s.c.) abolished it. The increases of PGE2 and ß-endorphin concentration in the CSF induced by AEA were abolished by the pretreatment of rats with AM251. These results suggest that endocannabinoids are intrinsically involved in the pyretic activity of cytokines (IL-1ß, TNF-α, IL-6), CRH and ET-1 but not the PGE2 or PGF2α induced fevers. However, anandamide via CB1 receptor activation induces fever that is dependent on the synthesis of prostaglandin and opioids.

Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol-Bound ß1-Adrenergic Receptor.

Comparisons between structures of the ß1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser(5.46), coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of ß1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both ß1AR and ß2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey ß1AR and an inverse agonist of human ß2AR. The structure of 7-methylcyanopindolol-bound ß1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound ß1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol-bound ß1AR and the hydroxyl group of Ser(5.46) is positioned 0.8 Å further from the ligand, with respect to the position of the Ser(5.46) side chain in cyanopindolol-bound ß1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.

Prevalence and management of chronic kidney disease in primary care patients in the UK.

BACKGROUND: This study aimed to estimate the prevalence of chronic kidney disease (CKD) in the UK in 2010 and to assess prevalence, comorbidities and comedications associated with the disease over time, following inclusion of CKD in the Quality and Outcomes Framework (QOF). METHODS: This was a retrospective, longitudinal study assessing individuals with prevalent or incident CKD (identified using estimated glomerular filtration rate readings and/or Read codes) in the General Practice Research Database (GPRD) in 2010. Individuals were assessed at two time points: in 2010 and at the date of their first classification of CKD in the GPRD. RESULTS: The prevalence of stage 3-5 CKD in 2010 was 5.9%. In patients with stage 3-5 CKD at first classification, their disease remained stable, progressed or improved by 2010 in approximately 50%, 10-15% and 25-30% of patients, respectively. Diagnoses of cardiovascular-related comorbidities (hypertension, hypercholesterolaemia, diabetes and cardiovascular disease), and treatment with antihypertensives and lipid-modifying therapy (LMT), increased with worsening disease severity. When patients were stratified by diagnosis date, the proportion of patients with stage 3-5 CKD and cardiovascular-related comorbidities decreased with time, and the relative use of LMT and antihypertensives among patients with hypercholesterolaemia and hypertension increased with time. CONCLUSIONS: Chronic kidney disease is generally stable or progressive, although more patients improve disease stage than previously assumed. Data suggest that the introduction of CKD into the QOF has increased awareness of CKD among physicians in the UK, allowing for earlier intervention and better control of CKD progression.

Impact of lipid-lowering therapy on the prevalence of dyslipidaemia in patients at high-risk of cardiovascular events in UK primary care - a retrospective database study.

AIMS: To estimate the prevalence of dyslipidaemias in high-risk patients new to lipid-modifying therapy (LMT), and establish the extent to which these lipid abnormalities are addressed by treatment in UK clinical practice. METHODS: The PRIMULA study was a retrospective analysis, conducted using the UK General Practice Research Database. Two periods were studied as follows: a pretreatment period, defined as the 12 months before initiation of LMT (the index date), and a follow-up period of at least 12 months. Patients included in the study (n = 25,011) had dyslipidaemia with at least one abnormal lipid measurement [total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) or triglycerides (TG)] in the pretreatment period. All patients were at high risk of cardiovascular events, which was defined as having a history of cardiovascular disease, a 10-year Framingham risk score higher than 20%, diabetes or hypertension, as defined by the Joint British Societies 2 guidelines. RESULTS: At the index date, 98% of patients were initiated on statin monotherapy. After 12 months of treatment, 15.2% (sub-group range: 11.0-22.9%) of all high-risk patients had no lipid abnormalities. The proportions of patients with high TC or LDL-C levels decreased from 98.8% to 68.9%, and from 99.2% to 68.7%, respectively, over 12 months. The prevalence of high TG levels decreased from 45.0% to 26.9%, whereas that of low HDL-C levels increased, from 16.6% to 18.0%. Risk factors for cardiovascular events were not consistently associated with the likelihood of attaining optimal lipid levels. CONCLUSIONS: Despite widespread use of statins, many individuals at high risk of cardiovascular events have persistently abnormal lipid levels, with over two-thirds of patients not achieving target levels of LDL-C or TC. Management of dyslipidaemia is therefore suboptimal in this important high-risk group in UK standard practice.

Point of Care CD4 Testing in National Household Surveys - Results and Quality Indicators from Eleven Population-Based HIV Impact Assessment (PHIA) Surveys.

Population-based HIV Impact Assessments (PHIAs) are national household (HH) surveys that provide HIV diagnosis and CD4 testing with an immediate return of results. Accurate CD4 results improve HIV-positive participants' clinical care and inform the effectiveness of HIV programs. Here, we present CD4 results from the PHIA surveys that were conducted in 11 countries in sub-Saharan Africa between 2015 and 2018. All of the HIV-positive participants and 2 to 5% of the HIV-negative participants were offered Pima CD4 (Abbott, IL, USA) point-of-care (POC) tests. The quality of the CD4 test was ensured by conducting instrument verification, comprehensive training, quality control, a review of testing errors and an analysis of unweighted CD4 data by HIV status, age, gender, and antiretroviral (ARV) treatment status. Overall, CD4 testing was completed for 23,085 (99.5%) of the 23,209 HIV-positive and 7,329 (2.7%) of the 270,741 negative participants in 11 surveys. The instrument error rate was 11.3% (range, 4.4% to 15.7%). The median CD4 values among HIV-positive and HIV-negative participants (aged 15+) were 468 cells/mm3 (interquartile range [IQR], 307 to 654) and 811 cells/mm3 (IQR, 647 to 1,013), respectively. Among the HIV-positive participants (aged 15+), those with detectable ARVs had higher CD4 values (508 cells/mm3) than those with undetectable ARVs (385.5 cells/mm3). Among the HIV-positive participants (aged 15+), 11.4% (2,528/22,253) had a CD4 value of less than 200 cells/mm3, and approximately half of them (1,225/2,528 = 48.5%) had detectable ARVs, whereas 51.5% (1,303/2,528) had no detectable ARVs (P < 0.0001). We successfully implemented high quality POC CD4 testing using Pima instruments. Our data come from nationally representative surveys in 11 countries and provide unique insights regarding the CD4 distribution among HIV-positive individuals as well as the baseline CD4 values among HIV-negative individuals. IMPORTANCE The manuscript describes CD4 levels among HIV-positive individuals and baseline CD4 levels among HIV-negative individuals from 11 sub-Saharan countries, thereby highlighting the importance of CD4 markers in the context of the HIV epidemic. Despite increased ARV access in each country, advanced HIV disease (CD4 < 200 cells/mm3) persists among approximately 11% of HIV-positive individuals. Therefore, it is important that our findings are shared with the scientific community to assist with similar implementations of point-of-care testing and to conduct a review of HIV programmatic gaps.

Microbubble and ultrasound radioenhancement of bladder cancer.

BACKGROUND: Tumour vasculature is an important component of tumour growth and survival. Recent evidence indicates tumour vasculature also has an important role in tumour radiation response. In this study, we investigated ultrasound and microbubbles to enhance the effects of radiation. METHODS: Human bladder cancer HT-1376 xenografts in severe combined immuno-deficient mice were used. Treatments consisted of no, low and high concentrations of microbubbles and radiation doses of 0, 2 and 8 Gy in short-term and longitudinal studies. Acute response was assessed 24 h after treatment and longitudinal studies monitored tumour response weekly up to 28 days using power Doppler ultrasound imaging for a total of 9 conditions (n=90 animals). RESULTS: Quantitative analysis of ultrasound data revealed reduced blood flow with ultrasound-microbubble treatments alone and further when combined with radiation. Tumours treated with microbubbles and radiation revealed enhanced cell death, vascular normalisation and areas of fibrosis. Longitudinal data demonstrated a reduced normalised vascular index and increased tumour cell death in both low and high microbubble concentrations with radiation. CONCLUSION: Our study demonstrated that ultrasound-mediated microbubble exposure can enhance radiation effects in tumours, and can lead to enhanced tumour cell death.

Why nanoprojectiles work differently than macroimpactors: the role of plastic flow.

Atomistic simulation data on crater formation due to the hypervelocity impact of nanoprojectiles of up to 55 nm diameter and with targets containing up to 1.1×10(10) atoms are compared to available experimental data on µm-, mm-, and cm-sized projectiles. We show that previous scaling laws do not hold in the nanoregime and outline the reasons: within our simulations we observe that the cratering mechanism changes, going from the smallest to the largest simulated scales, from an evaporative regime to a regime where melt and plastic flow dominate, as is expected in larger microscale experiments. The importance of the strain-rate dependence of strength and of dislocation production and motion are discussed.

Small understorey trees have greater capacity than canopy trees to adjust hydraulic traits following prolonged experimental drought in a tropical forest.

Future climate change predictions for tropical forests highlight increased frequency and intensity of extreme drought events. However, it remains unclear whether large and small trees have differential strategies to tolerate drought due to the different niches they occupy. The future of tropical forests is ultimately dependent on the capacity of small trees (<10 cm in diameter) to adjust their hydraulic system to tolerate drought. To address this question, we evaluated whether the drought tolerance of neotropical small trees can adjust to experimental water stress and was different from tall trees. We measured multiple drought resistance-related hydraulic traits across nine common neotropical genera at the world's longest-running tropical forest throughfall-exclusion experiment and compared their responses with surviving large canopy trees. Small understorey trees in both the control and the throughfall-exclusion treatment had lower minimum stomatal conductance and maximum hydraulic leaf-specific conductivity relative to large trees of the same genera, as well as a greater hydraulic safety margin (HSM), percentage loss of conductivity and embolism resistance, demonstrating that they occupy a distinct hydraulic niche. Surprisingly, in response to the drought treatment, small trees increased specific hydraulic conductivity by 56.3% and leaf:sapwood area ratio by 45.6%. The greater HSM of small understorey trees relative to large canopy trees likely enabled them to adjust other aspects of their hydraulic systems to increase hydraulic conductivity and take advantage of increases in light availability in the understorey resulting from the drought-induced mortality of canopy trees. Our results demonstrate that differences in hydraulic strategies between small understorey and large canopy trees drive hydraulic niche segregation. Small understorey trees can adjust their hydraulic systems in response to changes in water and light availability, indicating that natural regeneration of tropical forests following long-term drought may be possible.