The influence of backbone fluorination on the helicity of α/γ-hybrid peptides.

Peptides that are composed of an alternating pattern of α- and γ-amino acids are potentially valuable as metabolism-resistant bioactive agents. For optimal function, some kind of conformational restriction is usually required to either stabilize the dominant 12-helix, or else to divert the peptide away from this conformation in a controlled way. Herein, we explore stereoselective fluorination as a method for controlling the conformations of α/γ-hybrid peptides. We show through a combination of X-ray, NMR and CD analyses that fluorination can either stabilize or disrupt the 12-helix, depending on the fluorine stereochemistry. These findings could inform the ongoing development of diverse functional hybrid peptides.

Tinker, Tailor, Soldier, Spy: The Diverse Roles That Fluorine Can Play within Amino Acid Side Chains.

Side chain-fluorinated amino acids are useful tools in medicinal chemistry and protein science. In this review, we outline some general strategies for incorporating fluorine atom(s) into amino acid side chains and for elaborating such building blocks into more complex fluorinated peptides and proteins. We then describe the diverse benefits that fluorine can offer when located within amino acid side chains, including enabling 19F NMR and 18F PET imaging applications, enhancing pharmacokinetic properties, controlling molecular conformation, and optimizing target-binding.

Morpho-mechanistic screening criteria for the echocardiographic detection of rheumatic heart disease.

INTRODUCTION: Screening echocardiography, guided by the current World Heart Federation (WHF) criteria, has important limitations that impede the establishment of large-scale rheumatic heart disease (RHD) control programmes in endemic regions. The criteria misclassify a significant number of normal cases as borderline RHD. Prior attempts to simplify them are limited by incorporation bias due to the lack of an externally validated, accurate diagnostic test for RHD. We set out to assess novel screening criteria designed to avoid incorporation bias and to compare this against the performance of the current WHF criteria. METHODS: The performance of the WHF and the morpho-mechanistic (MM) RHD screening criteria (a novel set of screening criteria that evaluate leaflet morphology, motion and mechanism of regurgitation) as well as a simplified RHD MM 'rule-out' test (based on identifying a predefined sign of anterior mitral valve leaflet restriction for the mitral valve and any aortic regurgitation for the aortic valve) were assessed in two contrasting cohorts: first, a low-risk RHD cohort consisting of children with a very low-risk RHD profile. and second, a composite reference standard (CRS) RHD-positive cohort that was created using a composite of two criteria to ensure a cohort with the highest possible likelihood of RHD. Subjects included in this group required (1) proven, prior acute rheumatic fever and (2) current evidence of predefined valvular regurgitation on echocardiography. RESULTS: In the low-risk RHD cohort (n=364), the screening specificities for detecting RHD of the MM and WHF criteria were 99.7% and 95.9%, respectively (p=0.0002). The MM rule-out test excluded 359/364 cases (98.6%). In the CRS RHD-positive cohort (n=65), the screening sensitivities for the detection of definite RHD by MM and WHF criteria were 92.4% and 89.2%, respectively (p=0.2231). The MM RHD rule-out test did not exclude any cases from the CRS RHD-positive cohort. CONCLUSION: Our proposed MM approach showed an equal sensitivity to the WHF criteria but with significantly improved specificity. The MM RHD rule-out test excluded RHD-negative cases while identifying all cases within the CRS RHD-positive cohort. This holds promise for the development of a two-step RHD screening algorithm to enable task shifting in RHD endemic regions.

The potential and shortcomings of mitochondrial DNA analysis for cheetah conservation management.

There are only about 7,100 adolescent and adult cheetahs (Acinonyx jubatus) remaining in the wild. With the majority occurring outside protected areas, their numbers are rapidly declining. Evidence-based conservation measures are essential for the survival of this species. Genetic data is routinely used to inform conservation strategies, e.g., by establishing conservation units (CU). A commonly used marker in conservation genetics is mitochondrial DNA (mtDNA). Here, we investigated the cheetah's phylogeography using a large-scale mtDNA data set to refine subspecies distributions and better assign individuals to CUs. Our dataset mostly consisted of historic samples to cover the cheetah's whole range as the species has been extinct in most of its former distribution. While our genetic data largely agree with geography-based subspecies assignments, several geographic regions show conflicting mtDNA signals. Our analyses support previous findings that evolutionary forces such as incomplete lineage sorting or mitochondrial capture likely confound the mitochondrial phylogeography of this species, especially in East and, to some extent, in Northeast Africa. We caution that subspecies assignments solely based on mtDNA should be treated carefully and argue for an additional standardized nuclear single nucleotide polymorphism (SNP) marker set for subspecies identification and monitoring. However, the detection of the A. j. soemmeringii specific haplogroup by a newly designed Amplification-Refractory Mutation System (ARMS) can already provide support for conservation measures. Supplementary Information: The online version contains supplementary material available at 10.1007/s10592-022-01483-1.

Genomic analyses show extremely perilous conservation status of African and Asiatic cheetahs (Acinonyx jubatus).

We live in a world characterized by biodiversity loss and global environmental change. The extinction of large carnivores can have ramifying effects on ecosystems like an uncontrolled increase in wild herbivores, which in turn can have knock-on impacts on vegetation regeneration and communities. Cheetahs (Acinonyx jubatus) serve important ecosystem functions as apex predators; yet, they are quickly heading towards an uncertain future. Threatened by habitat loss, human-wildlife conflict and illegal trafficking, there are only approximately 7100 individuals remaining in nature. We present the most comprehensive genome-wide analysis of cheetah phylogeography and conservation genomics to date, assembling samples from nearly the entire current and past species' range. We show that their phylogeography is more complex than previously thought, and that East African cheetahs (A. j. raineyi) are genetically distinct from Southern African individuals (A. j. jubatus), warranting their recognition as a distinct subspecies. We found strong genetic differentiation between all classically recognized subspecies, thus refuting earlier findings that cheetahs show only little differentiation. The strongest differentiation was observed between the Asiatic and all the African subspecies. We detected high inbreeding in the Critically Endangered Iranian (A. j. venaticus) and North-western (A. j. hecki) subspecies, and show that overall cheetahs, along with snow leopards, have the lowest genome-wide heterozygosity of all the big cats. This further emphasizes the cheetah's perilous conservation status. Our results provide novel and important information on cheetah phylogeography that can support evidence-based conservation policy decisions to help protect this species. This is especially relevant in light of ongoing and proposed translocations across subspecies boundaries, and the increasing threats of illegal trafficking.

Fluorinated quorum sensing inhibitors: enhancement of potency through conformational control.

The bacterial quorum sensing (QS) system is a target for non-lethal antibacterial agents that do not encourage the development of resistance. QS inhibitors commonly contain a polar "head" moiety and a lipidic "tail" moiety. In this work, we synthesised novel QS inhibitor candidates in which the lipidic "tail" is decorated with stereospecifically positioned fluorine atoms. The presence of fluorine is shown to bias the molecules into distinctive conformations that are pre-organised for binding to the QS receptor. This translates into significant increases in QS inhibitory potency.

The Effect of Vicinal Difluorination on the Conformation and Potency of Histone Deacetylase Inhibitors.

Histone deacetylase enzymes (HDACs) are potential targets for the treatment of cancer and other diseases, but it is challenging to design isoform-selective agents. In this work, we created new analogs of two established but non-selective HDAC inhibitors. We decorated the central linker chains of the molecules with specifically positioned fluorine atoms in order to control the molecular conformations. The fluorinated analogs were screened against a panel of 11 HDAC isoforms, and minor differences in isoform selectivity patterns were observed.

Discovery of neuroprotective agents that inhibit human prolyl hydroxylase PHD2.

Prolyl hydroxylase (PHD) enzymes play a critical role in the cellular responses to hypoxia through their regulation of the hypoxia inducible factor α (HIF-α) transcription factors. PHD inhibitors show promise for the treatment of diseases including anaemia, cardiovascular disease and stroke. In this work, a pharmacophore-based virtual high throughput screen was used to identify novel potential inhibitors of human PHD2. Two moderately potent new inhibitors were discovered, with IC50 values of 4 µM and 23 µM respectively. Cell-based studies demonstrate that these compounds exhibit protective activity in neuroblastoma cells, suggesting that they have the potential to be developed into clinically useful neuroprotective agents.

The variable spectrum of anterior mitral valve leaflet restriction in rheumatic heart disease screening.

INTRODUCTION: The World Heart Federation (WHF) screening criteria do not incorporate a strict, reproducible definition of anterior mitral valve leaflet (AMVL) restriction. Using a novel definition, we have identified two distinct AMVL restriction configurations. The first, called "distal tip" AMVL restriction is associated with additional morphological features of rheumatic heart disease (RHD), while the second, "gradual bowing" AMVL restriction is not. This "arch-like" leaflet configuration involves the base to tip of the medial MV in isolation. We hypothesize that this configuration is a normal variant. METHODOLOGY: The prevalence and associated leaflet configurations of AMVL restriction were assessed in schoolchildren with an established "very low" (VLP), "high" (HP), and "very high" prevalence (VHP) of RHD. RESULTS: 936 studies were evaluated (HP 577 cases; VLP 359 cases). Sixty-five cases of "gradual bowing" AMVL restriction were identified in the HP cohort (11.3%, 95% CI 8.9-14.1) and 35 cases (9.7%, 95% CI 7-13.2) in the VLP cohort (P = .47). In the second analyses, an enriched cohort of 43 studies with proven definite RHD were evaluated. "Distal tip" AMVL restriction was identified in all 43 VHP cases (100%) and affected the central portion of the AMVL in all cases. CONCLUSION: "Gradual bowing" AMVL restriction appears to be a normal, benign variant of the MV, not associated with RHD risk nor with any other morphological features of RHD. Conversely, "Distal tip" AMVL restriction was present in all cases in the VHP cohort with no cases exhibiting a straight, nonrestricted central portion of the AMVL. This novel finding requires further investigation as a potential RHD rule-out test of the MV.

Screening for subclinical rheumatic heart disease: addressing borderline disease in a real-world setting.

Aims: The World Heart Federation (WHF) criteria identify a large borderline rheumatic heart disease (RHD) category that has hampered the implementation of population-based screening. Inter-scallop separations (ISS) of the posterior mitral valve leaflet, a recently described normal variant of the mitral valve, appears to be an important cause of mild mitral regurgitation (MR) leading to misclassification of cases as WHF 'borderline RHD'. This study aims to report the findings of the Echo in Africa project, a large-scale RHD screening project in South Africa and determine what proportion of borderline cases would be re-classified as normal if there were a systematic identification of ISS-related MR. Methods and results: A prospective cross-sectional study of underserved secondary schools in the Western Cape was conducted. Participants underwent a screening study with a handheld (HH) ultrasound device. Children with an abnormal HH study were re-evaluated with a portable laptop echocardiography machine. A mechanistic evaluation was applied in cases with isolated WHF 'pathological' MR (WHF 'borderline RHD'). A total of 5255 participants (mean age 15± years) were screened. A total of 3439 (65.8%) were female. Forty-nine cases of WHF 'definite RHD' [9.1 cases/1000 (95% confidence interval, CI, 6.8-12.1 cases/1000)] and 104 cases of WHF 'borderline RHD' [19.5 cases/1000 (95% CI, 16.0-23.7 cases/1000)] were identified. Inter-scallop separations-related MR was the underlying mechanism of MR in 48/68 cases classified as WHF 'borderline RHD' with isolated WHF 'pathological' MR (70.5%). Conclusion: In a real-world, large-scale screening project, the adoption of a mechanistic evaluation based on the systematic identification of ISS-related MR markedly reduced the number of WHF 'screen-positive' cases misclassified as WHF 'borderline RHD'. Implementing strategies that reduce this misclassification could reduce the cost- and labour burden on large-scale RHD screening programmes.

Vicinal difluorination as a C=C surrogate: an analog of piperine with enhanced solubility, photostability, and acetylcholinesterase inhibitory activity.

Piperine, a natural product derived from peppercorns, has a variety of biological activities that make it an attractive lead compound for medicinal chemistry. However, piperine has some problematic physicochemical properties including poor aqueous solubility and a susceptibility to UV-induced degradation. In this work, we designed an analog of piperine in which the central conjugated hydrocarbon chain is replaced with a vicinal difluoroalkane moiety. We show that this fluorinated analog of piperine has superior physicochemical properties, and it also has higher potency and selectivity towards one particular drug target, acetylcholinesterase. This work highlights the potential usefulness of the threo-difluoroalkane motif as a surrogate for E-alkenes in medicinal chemistry.

Interscallop separations of the posterior mitral valve leaflet: a solution to the 'borderline RHD' conundrum?

OBJECTIVE: The World Heart Federation (WHF) criteria incorporate a Doppler-based system to differentiate between 'physiological' and 'pathological' mitral regurgitation (MR)-a sole criterion sufficient for the diagnosis of WHF 'borderline' rheumatic heart disease (RHD). We have identified that interscallop separations (ISS) of the posterior mitral valve (MV) leaflet, can give rise to pathological MR in an otherwise-normal MV. We aimed to establish and compare the prevalence of ISS-related MR among South African children at high and low risk for RHD. METHODS: A prospective cross-sectional echocardiographic study of 759 school children (aged 13-18) was performed. Cases with MR≥1.5 cm underwent a second comprehensive study to determine the prevalence of RHD according to the WHF guideline and establish the underlying mechanism of MR. RESULTS: Of 400 high-risk children, two met criteria for 'definite RHD' (5 per 1000 (95% CI 1.4 to 18.0); p=0.5) and 11 for 'borderline RHD' (27.5 per 1000 (95% CI 15.4 to 48.6)). Of 359 low-risk children, 14 met criteria for borderline RHD (39 per 1000 (95% CI 23.4 to 64.4)). Comprehensive echocardiography identified an underlying ISS as the mechanism of isolated pathological MR in 10 (83.3%) high-risk children and 11 low-risk children (78.5%; p>0.99). CONCLUSIONS: ISS are a ubiquitous finding among South African schoolchildren from all risk profiles and are regularly identified as the underlying mechanism of WHF pathological MR in borderline RHD cases. A detailed MV assessment with an emphasis on ascertaining the underlying mechanism of dysfunction could reduce the reported numbers of screened cases misclassified as borderline RHD.

Diastereoselective synthesis and conformational analysis of 4,5-difluoropipecolic acids.

Stereoselectively-fluorinated analogs of pipecolic acid have been investigated through a combined theoretical and experimental approach. Three of the four possible diastereoisomers of 4,5-difluoropipecolic acid were successfully synthesized via deoxyfluorination chemistry, navigating a complex reaction network that included neighboring group participation, rearrangement, and elimination pathways. A DFT-based conformational study, supported by NMR J-based analysis, revealed that the different diastereoisomers of 4,5-difluoropipecolic acid preferentially adopt different puckers of the six-membered ring. These findings could have future relevance for the conformational control of biologically active peptides.

Habitat complexity and lifetime predation risk influence mesopredator survival in a multi-predator system.

Variability in habitat selection can lead to differences in fitness; however limited research exists on how habitat selection of mid-ranking predators can influence population-level processes in multi-predator systems. For mid-ranking, or mesopredators, differences in habitat use might have strong demographic effects because mesopredators need to simultaneously avoid apex predators and acquire prey. We studied spatially-explicit survival of cheetahs (Acinonyx jubatus) in the Mun-Ya-Wana Conservancy, South Africa, to test hypotheses related to spatial influences of predation risk, prey availability, and vegetation complexity, on mesopredator survival. For each monitored cheetah, we estimated lion encounter risk, prey density, and vegetation complexity within their home range, on short-term (seasonal) and long-term (lifetime) scales and estimated survival based on these covariates. Survival was lowest for adult cheetahs and cubs in areas with high vegetation complexity on both seasonal and lifetime scales. Additionally, cub survival was negatively related to the long-term risk of encountering a lion. We suggest that complex habitats are only beneficial to mesopredators when they are able to effectively find and hunt prey, and show that spatial drivers of survival for mesopredators can vary temporally. Collectively, our research illustrates that individual variation in mesopredator habitat use can scale-up and have population-level effects.

Strategies for Fine-Tuning the Conformations of Cyclic Peptides.

Cyclic peptides are promising scaffolds for drug development, attributable in part to their increased conformational order compared to linear peptides. However, when optimizing the target-binding or pharmacokinetic properties of cyclic peptides, it is frequently necessary to "fine-tune" their conformations, e.g., by imposing greater rigidity, by subtly altering certain side chain vectors, or by adjusting the global shape of the macrocycle. This review systematically examines the various types of structural modifications that can be made to cyclic peptides in order to achieve such conformational control.

Screening for rheumatic heart disease: The reliability of anterior mitral valve leaflet thickness measurement.

BACKGROUND: Studies determining the reliability of the World Heart Federation (WHF) anterior mitral valve leaflet (AMVL) measurement are limited by the introduction of bias in their test-retest analyses. This study sought to determine the reliability of the current AMVL measurement while controlling for systematic bias. METHODS: Retrospective analysis of echocardiographic data from 16 patients with previous acute rheumatic fever was performed. Included in this study was an optimized cine loop of the mitral valve (MV) [reader-optimized measurement (ROM]) in the parasternal long-axis view and an optimized still image of the MV obtained from the same cine loop [specialist-optimized image (SOI)]. Each still image and associated cine loop was quadruplicated and randomized to determine intra- and inter-rater agreement and quantify the impact of zoom on AMVL measurement. RESULTS: Specialist-optimized image without zoom reflected the highest degree of agreement in both cohorts with an ICC of 0.29 and 0.46. The agreement in ROM images without zoom was ICC of 0.23 and 0.45. The addition of zoom to SOI decreased agreement further to an ICC of 0.20 and 0.36. The setting associated with the poorest agreement profile was ROI with zoom with an ICC of 0.13 and 0.34, respectively. The intra-rater agreement between readers in both cohorts was moderate across all settings with an ICC ranging between 0.64 and 0.86. CONCLUSIONS: The WHF AMVL measurement is only moderately repeatable within readers and demonstrates poor reproducibility that was not improved by the addition of a zoom-optimized protocol. Given our study findings, we cannot advocate the current WHF AMVL measurement as a reliable assessment for RHD.

Revisiting Inflammatory Bowel Disease: Pathology, Treatments, Challenges and Emerging Therapeutics Including Drug Leads from Natural Products.

Inflammatory bowel disease (IBD) is a chronic and life-long disease characterized by gastrointestinal tract inflammation. It is caused by the interplay of the host's genetic predisposition and immune responses, and various environmental factors. Despite many treatment options, there is no cure for IBD. The increasing incidence and prevalence of IBD and lack of effective long-term treatment options have resulted in a substantial economic burden to the healthcare system worldwide. Biologics targeting inflammatory cytokines initiated a shift from symptomatic control towards objective treatment goals such as mucosal healing. There are seven monoclonal antibody therapies excluding their biosimilars approved by the US Food and Drug Administration for induction and maintenance of clinical remission in IBD. Adverse side effects associated with almost all currently available drugs, especially biologics, is the main challenge in IBD management. Natural products have significant potential as therapeutic agents with an increasing role in health care. Given that natural products display great structural diversity and are relatively easy to modify chemically, they represent ideal scaffolds upon which to generate novel therapeutics. This review focuses on the pathology, currently available treatment options for IBD and associated challenges, and the roles played by natural products in health care. It discusses these natural products within the current biodiscovery research agenda, including the applications of drug discovery techniques and the search for next-generation drugs to treat a plethora of inflammatory diseases, with a major focus on IBD.

Unsustainable anthropogenic mortality disrupts natal dispersal and promotes inbreeding in leopards.

Anthropogenic mortality of wildlife is typically inferred from measures of the absolute decline in population numbers. However, increasing evidence suggests that indirect demographic effects including changes to the age, sex, and social structure of populations, as well as the behavior of survivors, can profoundly impact population health and viability. Specifically, anthropogenic mortality of wildlife (especially when unsustainable) and fragmentation of the spatial distribution of individuals (home-ranges) could disrupt natal dispersal mechanisms, with long-term consequences to genetic structure, by compromising outbreeding behavior and gene flow. We investigate this threat in African leopards (Panthera pardus pardus), a polygynous felid with male-biased natal dispersal. Using a combination of spatial (home-range) and genetic (21 polymorphic microsatellites) data from 142 adult leopards, we contrast the structure of two South African populations with markedly different histories of anthropogenically linked mortality. Home-range overlap, parentage assignment, and spatio-genetic autocorrelation together show that historical exploitation of leopards in a recovering protected area has disrupted and reduced subadult male dispersal, thereby facilitating opportunistic male natal philopatry, with sons establishing territories closer to their mothers and sisters. The resultant kin-clustering in males of this historically exploited population is comparable to that of females in a well-protected reserve and has ultimately led to localized inbreeding. Our findings demonstrate novel evidence directly linking unsustainable anthropogenic mortality to inbreeding through disrupted dispersal in a large, solitary felid and expose the genetic consequences underlying this behavioral change. We therefore emphasize the importance of managing and mitigating the effects of unsustainable exploitation on local populations and increasing habitat fragmentation between contiguous protected areas by promoting in situ recovery and providing corridors of suitable habitat that maintain genetic connectivity.

Understanding decision making in a food-caching predator using hidden Markov models.

BACKGROUND: Tackling behavioural questions often requires identifying points in space and time where animals make decisions and linking these to environmental variables. State-space modeling is useful for analysing movement trajectories, particularly with hidden Markov models (HMM). Yet importantly, the ontogeny of underlying (unobservable) behavioural states revealed by the HMMs has rarely been verified in the field. METHODS: Using hidden Markov models of individual movement from animal location, biotelemetry, and environmental data, we explored multistate behaviour and the effect of associated intrinsic and extrinsic drivers across life stages. We also decomposed the activity budgets of different movement states at two general and caching phases. The latter - defined as the period following a kill which likely involves the caching of uneaten prey - was subsequently confirmed by field inspections. We applied this method to GPS relocation data of a caching predator, Persian leopard Panthera pardus saxicolor in northeastern Iran. RESULTS: Multistate modeling provided strong evidence for an effect of life stage on the behavioural states and their associated time budget. Although environmental covariates (ambient temperature and diel period) and ecological outcomes (predation) affected behavioural states in non-resident leopards, the response in resident leopards was not clear, except that temporal patterns were consistent with a crepuscular and nocturnal movement pattern. Resident leopards adopt an energetically more costly mobile behaviour for most of their time while non-residents shift their behavioural states from high energetic expenditure states to energetically less costly encamped behaviour for most of their time, which is likely to be a risk avoidance strategy against conspecifics or humans. CONCLUSIONS: This study demonstrates that plasticity in predator behaviour depending on life stage may tackle a trade-off between successful predation and avoiding the risks associated with conspecifics, human presence and maintaining home range. Range residency in territorial predators is energetically demanding and can outweigh the predator's response to intrinsic and extrinsic variables such as thermoregulation or foraging needs. Our approach provides an insight into spatial behavior and decision making of leopards, and other large felids in rugged landscapes through the application of the HMMs in movement ecology.