A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain.

Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs-a long ncRNA, a circular RNA, and two microRNAs-using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more effective than previously described examples of target-directed microRNA degradation, which come primarily from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as in neurons, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network.

MicroRNA turnover: a tale of tailing, trimming, and targets.

MicroRNAs (miRNAs) post-transcriptionally repress gene expression by guiding Argonaute (AGO) proteins to target mRNAs. While much is known about the regulation of miRNA biogenesis, miRNA degradation pathways are comparatively poorly understood. Although miRNAs generally exhibit slow turnover, they can be rapidly degraded through regulated mechanisms that act in a context- or sequence-specific manner. Recent work has revealed a particularly important role for specialized target interactions in controlling rates of miRNA degradation. Engagement of these targets is associated with the addition and removal of nucleotides from the 3' ends of miRNAs, a process known as tailing and trimming. Here we review these mechanisms of miRNA modification and turnover, highlighting the contexts in which they impact miRNA stability and discussing important questions that remain unanswered.

Structural Basis for Target-Directed MicroRNA Degradation.

MicroRNAs (miRNAs) broadly regulate gene expression through association with Argonaute (Ago), which also protects miRNAs from degradation. However, miRNA stability is known to vary and is regulated by poorly understood mechanisms. A major emerging process, termed target-directed miRNA degradation (TDMD), employs specialized target RNAs to selectively bind to miRNAs and induce their decay. Here, we report structures of human Ago2 (hAgo2) bound to miRNAs and TDMD-inducing targets. miRNA and target form a bipartite duplex with an unpaired flexible linker. hAgo2 cannot physically accommodate the RNA, causing the duplex to bend at the linker and display the miRNA 3' end for enzymatic attack. Altering 3' end display by changing linker flexibility, changing 3' end complementarity, or mutationally inducing 3' end release impacts TDMD efficiency, leading to production of distinct 3'-miRNA isoforms in cells. Our results uncover the mechanism driving TDMD and reveal 3' end display as a key determinant regulating miRNA activity via 3' remodeling and/or degradation.

Tailer: a pipeline for sequencing-based analysis of nonpolyadenylated RNA 3' end processing.

Post-transcriptional trimming and tailing of RNA 3' ends play key roles in the processing and quality control of noncoding RNAs (ncRNAs). However, bioinformatic tools to examine changes in the RNA 3' "tailome" are sparse and not standardized. Here we present Tailer, a bioinformatic pipeline in two parts that allows for robust quantification and analysis of tail information from next-generation sequencing experiments that preserve RNA 3' end information. The first part of Tailer, Tailer-processing, uses genome annotation or reference FASTA gene sequences to quantify RNA 3' ends from SAM-formatted alignment files or FASTQ sequence read files produced from sequencing experiments. The second part, Tailer-analysis, uses the output of Tailer-processing to identify statistically significant RNA targets of trimming and tailing and create graphs for data exploration. We apply Tailer to RNA 3' end sequencing experiments from three published studies and find that it accurately and reproducibly recapitulates key findings. Thus, Tailer should be a useful and easily accessible tool to globally investigate tailing dynamics of nonpolyadenylated RNAs and conditions that perturb them.

How to use CRISPR/Cas9 in plants - from target site selection to DNA repair.

A tool for precise, target-specific, efficient and affordable genome editing, it is a dream for many researchers, from those who do basic research to those who use it for applied research. Since 2012, we have the tool that almost fulfils such requirements; it is based on CRISPR/Cas systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight the key factors affecting its mutagenesis outcomes. Firstly, factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state or Cas9 variant, and how long it remains in place after cleavage. Secondly, factors affecting the follow-up DNA repair mechanisms include mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

Bovine Digital Dermatitis: Treponema spp. on trimming equipment and chutes - effect of washing and disinfection.

BACKGROUND: Digital dermatitis (DD) is a contagious bovine foot disease causing reduced animal welfare and negative economic consequences for the farmer. Treponema spp. are the most important causative agents. Studies indicate that trimming equipment can transfer DD-associated treponemes between cows. The aim of this observational study in 22 DD-positive Norwegian dairy herds was to investigate the risk of transferring Treponema spp. with trimming equipment and chutes after claw trimming, and after washing and disinfection. Swabs from the trimming equipment and chutes were collected from nine different locations, at five different time points. Bacterial DNA was extracted from 647 swabs and analysed by qPCR for Treponema spp. In addition, 172 swabs taken immediately after trimming, were analysed by a multiplex qPCR targeting T. phagedenis, T. pedis and T. medium/vincentii. Biopsy sampling from DD lesions was performed on cows in the same herds during trimming. Altogether 109 biopsies were analysed by FISH for confirmation of the DD diagnosis and identification of Treponema phylotypes (PTs). RESULTS: High numbers of Treponema spp. were detected from all nine locations on the trimming equipment and chutes immediately after trimming, and T. phagedenis was detected on two or more locations in all but two herds, 1 and 19. There was a decline in the amount of Treponema spp. after washing and disinfection. The belly belt, the cuff, and the footrest on the chute had the highest proportion of positive samples after disinfection. The belly belt had the highest copy numbers of all nine locations (median = 7.9, max = 545.1). No Treponema spp. was detected on the hoof knives after disinfection. Treponema phagedenis, T. pedis, and Treponema phylotype 3 (T. refringens) were detected by FISH analysis of the biopsies. Treponema phagedenis was detected in biopsies from all herds except 1 and 19. CONCLUSION: This study shows that DD-associated Treponema spp. were present on the trimming equipment and chutes after trimming cows in DD-positive herds. Washing and disinfection reduced the load of Treponema spp. However, large differences in Treponema spp. between different locations were documented. High copy numbers on the grinder and the chute after disinfection, indicates that sufficient cleaning and disinfection of these locations is difficult, and that passive transfer of DD-associated treponemes (viable or not) is possible.

An abattoir study of the prevalence of foot lesions and claw measurements in water buffalo in Egypt.

BACKGROUND: Lameness has been associated with compromised animal welfare and reduced productivity in dairy cattle herds worldwide. However, little is known about the prevalence of claw lesions in the dairy buffalo population in Egypt. Furthermore, the optimum measurements for claw trimming in buffalo are unknown. A cross-sectional cadaver study was conducted where 135 pair buffalo hind feet were collected from 4 slaughterhouses and examined for the presence of claw lesions. The proportion and associated 95% confidence interval (CI) of each type of lesion were calculated. A separate set of healthy claws (n = 26) underwent ultrasonography (US) and computed tomography (CT). The agreement between US and CT measurements was assessed using Passing-Bablok regression and intraclass correlation coefficient. The CT measurements were used to calculate trimming recommendations. RESULTS: At least one lesion was identified in 242 claws (89.6%, 95% CI = 85.4-93.0). In healthy claws, poor to moderate agreement was identified between US and CT measurements which could be due a sample size of the study. The average ± standard deviation (SD) minimum recommended external wall length of the lateral and medial claws in heifers was 7.1 ± 0.36 cm and 7.5 ± 0.35 cm, respectively. The average ± SD minimum recommended external wall length in buffaloes over five years of age was 8.2 ± 0.27 cm and 8.4 ± 0.39 cm for the lateral and medial claws, respectively. CONCLUSIONS: The study found a high prevalence of claw lesions in buffalo in Egypt, the clinical significance of which requires further elucidation. Recommended measurements will help guide claw trimming in buffalo to minimise lesions.

Photogrammetric analysis of limb joint angles in normal-gait cows before and after hoof trimming.

The aim of this study was to evaluate the effect of hoof trimming on overall limb movements by comparing the changes in 8 limb joint angles before and after one week of hoof trimming. Seventeen Holstein-Friesian dairy cows that were able to move freely and had no history of hoof diseases were included in the study. The cows were walked on a rubber mat with a high friction coefficient (HFM) and a low friction coefficient by the spraying of sodium polyacrylate (LFM). A high-speed camera was set to 200 fps on the image analysis software, and the images of the cows that were given 15 reflective markers on their right side were captured while walking on the test mat. The tests were conducted before and after one week of hoof trimming, and the cows were trimmed by the functional hoof trimming method. With image analysis software, video clips of walking cows were confirmed visually and tracked during one gait cycle by each reflective marker attached to the hoof of the forelimb and hindlimb, after which the stance phase and swing phase were identified. The durations of the stance phase and swing phase of the forelimb and hindlimb, respectively, and the maximum, minimum, and range of motion (ROM) values of the 8 joint angles, shoulder joint, elbow joint, carpus joint, forelimb fetlock joint, hip joint, stifle joint, hock joint and hindlimb fetlock joint during one gait cycle were included in the analysis. The maximum and minimum angles of the hip and stifle joints were narrower after hoof trimming than before, although the ROM did not change and was clearer for HFM than for LFM. It was thought that the flexion of the proximal hindlimb would progress smoothly during walking after trimming.

Association between hoof trimming frequency in primiparous cows and hoof health and survival in second lactation.

Lameness, mostly resulting from claw lesions, causes major welfare problems in the dairy industry. One way to prevent claw lesions is hoof trimming, but the overall effect and the optimal hoof trimming frequency are unknown. In a retrospective cohort study, we investigated the association between hoof trimming frequency in primiparous cows and hoof health and culling in second lactation. We based our analysis on breed, calving and culling data in the period 2015-2018 for 30,613 primiparous cows in 202 Swedish dairy herds. Of the cows enrolled, 20% were not hoof-trimmed, 30% were trimmed once, 34% were trimmed twice, 13% were trimmed 3 times, and 2% were trimmed ≥ 4 times. We used multivariable mixed-effect logistic and multivariable multinomial logistic regression models to investigate the association between trimming frequency and 2 outcomes: hoof lesions at first trimming occasion within 90 d from second calving and culling in second lactation within 300 d after second calving. In general, cows trimmed 2 or 3 times during first lactation were at lower odds of having claw lesions compared with cows that were not trimmed (Odds Ratio (OR) = 0.66, 95% CI = 0.62-0.71 and 0.60, 95% CI = 0.55-0.66, respectively) or cows trimmed once (OR = 0.80, 95% CI = 0.75-0.85 and OR = 0.72, 95% CI = 0.66-0.79, respectively), and this beneficial effect was observed for most types of claw lesions. Moreover, cows trimmed 2, 3, or ≥ 4 times were at lower odds of being culled compared with cows that were not trimmed (OR = 0.71, 95% CI = 0.65-0.77, 0.68, 95% CI = 0.61-0.76 and 0.70, 95% CI = 0.54-0.90, respectively) or trimmed once (OR = 0.77, 95% CI = 0.71-0.83, OR = 0.74, 95% CI = 0.66-0.82 and OR = 0.75, 95% CI = 0.59-0.97, respectively). In particular, 2 trimmings, compared with 1 or no trimming, lowered the relative risk-ratio of dying or being euthanized on-farm, or being culled due to claw and leg disorders. More than 1 hoof trimming in first lactation also reduced the relative-risk ratio of being culled for other reasons. In conclusion, 2 or 3 hoof trimmings during first lactation were generally beneficial for hoof health in early second lactation and survival in second lactation. These findings can help improve animal welfare and production by reducing claw lesions, and thereby lameness among dairy cows, which would increase the longevity of dairy cows and the sustainability of the dairy industry.

Conformational Changes and Unfolding of ß-Amyloid Substrates in the Active Site of γ-Secretase.

Alzheimer's disease (AD) is the leading cause of dementia and is characterized by a presence of amyloid plaques, composed mostly of the amyloid-ß (Aß) peptides, in the brains of AD patients. The peptides are generated from the amyloid precursor protein (APP), which undergoes a sequence of cleavages, referred as trimming, performed by γ-secretase. Here, we investigated conformational changes in a series of ß-amyloid substrates (from less and more amyloidogenic pathways) in the active site of presenilin-1, the catalytic subunit of γ-secretase. The substrates are trimmed every three residues, finally leading to Aß40 and Aß42, which are the major components of amyloid plaques. To study conformational changes, we employed all-atom molecular dynamics simulations, while for unfolding, we used steered molecular dynamics simulations in an implicit membrane-water environment to accelerate changes. We have found substantial differences in the flexibility of extended C-terminal parts between more and less amyloidogenic pathway substrates. We also propose that the positively charged residues of presenilin-1 may facilitate the stretching and unfolding of substrates. The calculated forces and work/energy of pulling were exceptionally high for Aß40, indicating why trimming of this substrate is so infrequent.

Complications after CyPass® Micro-Stent explantation: a case series.

PURPOSE: To describe the in toto explantation of the CyPass® Micro-Stent and its conceivable complications. METHODS: This is a case series of eighteen eyes from fourteen patients who underwent CyPass® Micro-Stent implantation due to mild to moderate glaucoma and who subsequently suffered from loss of endothelial cell density. Consequently, the CyPass® Micro-Stent was in toto explanted. The surgical procedure and its complications are described and compared with trimming of the CyPass® Micro-Stent. RESULTS: A postoperative hyphema was developed in 8 of the 18 eyes. In four of them the hyphema was self-limiting, while in two patients an anterior chamber irrigation was necessary. One patient suffered from a severe intracameral bleeding and iridodialysis during explantation, so that the base of the iris had to be scleral fixated. The remaining explantations were without complications. CONCLUSION: Dealing with implanted CyPass® Micro-Stents poses a challenge for ophthalmic surgeons. An in toto removal can be traumatic, since the CyPass stent often is fibrotic encapsulated and fused with the surrounding tissue. Alternatively, trimming of the CyPass is also a viable option to avoid further endothelial damage. Reported complications of CyPass trimming are consistent with those that can occur after explantation. Further data on the development of the endothelial cells after trimming or explantation are not yet available. Therefore, it remains open whether trimming of the CyPass, in contrast to complete removal, carries the risk of further endothelial cell loss.

Long and short whiskers differently guide snout/pellet interaction in rat oral grasping.

We studied the role of rat whisker/snout tactile sense during oral grasping, comparing control data with those obtained, respectively, 1-3 and 5-7 days after bilateral long or short whisker trimming and 3-5 and 8-10 days after bilateral infraorbital nerve (ION) severing. Two behavioural phases were identified: whisker-snout contact by nose-N or lip-L and snout-tongue contact. The second phase involved either: snout passing over stationary pellet (Still pellet); pellet rolling as the snout passed over it (Rolling pellet); pellet being pushed forward by the snout (Pushed pellet); or pellet being hit and pushed away (Hit/Lost pellet). In controls, success was 100%, with N-contact prevailing over L-contact in the first phase and Still pellet in the second. In long whisker-trimmed versus controls, success was still 100%, but L-contact increased in frequency, Pushed pellet prevailed and the second phase duration increased. In short whisker-trimmed versus controls, success remained 100%, with increased L-contact frequency; the first phase duration did not change, but the second phase increased since in pushed trials, the pellet rolled around the snout. In ION-severed versus controls, both phases changed drastically: L-contact frequency increased, Pushed pellet prevailed and contact was persistently maintained; Hit/Lost pellet emerged, Still and Rolling pellets disappeared and the oral-grasping sequence was not triggered. These results suggest that long and short whiskers, respectively, optimize the first and second phases of snout-pellet interaction and that whisker/snout sense is necessary to trigger oral grasping. Kinematic trajectory analysis supports the conclusion that movement from whisker to snout contact is an orientation response.

Coupled folding-upon-binding of human tumor suppressor MIG6 to lung cancer EGFR kinase domain and molecular trimming/stapling of MIG6-derived ß-hairpins to target the coupling event.

Human epidermal growth factor receptor (EGFR) is involved in strong association with malignant proliferation, which has been shown to play a central role in the development and progression of non-small cell lung cancer and other solid tumors. The tumor-suppressor protein MIG6 is a negative regulator of EGFR kinase activity by binding at the activation interface of asymmetric dimer of EGFR kinase domain to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two discrete fragments 1 and 2 to directly interact with EGFR. It is revealed that the MIG6 fragment 2 is intrinsically disordered in free unbound state, but would fold into a well-structured ß-hairpin when binding to EGFR, thus characterized by a so-called coupled folding-upon-binding process, which can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, a 23-mer F2P peptide was derived from MIG6 fragment 2, trimmed into a 17-mer tF2P peptide that contains the binding hotspot region of the fragment 2, and then constrained with an ordered hairpin conformation in free unbound state by disulfide stapling, finally resulting in a rationally stapled/trimmed stF2P peptide that largely minimizes the unfavorable indirect readout effect upon its binding to EGFR kinase domain, with affinity improved considerably upon the trimming and stapling/trimming. These rationally designed ß-hairpin peptides may be further exploited as potent anti-lung cancer agents to target the activation event of EGFR dimerization.

Structure-function analysis of microRNA 3'-end trimming by Nibbler.

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/ß-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.

Comparative study of trimming and resection of diseased finger superficial flexor tendons in gouty carpal tunnel syndrome.

BACKGROUND: Hyperuricemia can lead to synovial hyperplasia in the wrist. In severe cases, it can lead to the deposition of gouty stone in the carpal tunnel, resulting in increased pressure in the carpal tunnel and compression of the median nerve to cause carpal tunnel syndrome (CTS), which is called gouty carpal tunnel syndrome (GCTS). As for the surgical treatment of gouty carpal tunnel syndrome, scholars have different opinions on whether it is necessary to remove the superficial flexor tendon. The purpose of this study was to compare the clinical efficacy of trimming and resection of the diseased superficial flexor tendon in the treatment of gouty carpal tunnel syndrome. METHODS: Clinical data were collected from May 2016 to July 2021 from 10 patients (13 affected wrists) diagnosed with gouty carpal tunnel syndrome and classified into two groups according to the surgical modality: the diseased portion of the gout-eroded superficial finger tendon was trimmed in 9 wrists, and the diseased superficial finger flexor tendon was excised in 4 wrists. Values related to flexion and extension functions, 2-PD, DASH, BCTQ, VAS and recurrence in the affected fingers were compared between the two groups as well as before and after surgery in each group. RESULTS: All affected limbs used were cleared of gouty stones, finger numbness improved, no skin necrosis occurred, and all incisions healed at stage I. At follow-up (13.58 ± 5.53 months), there was no significant difference between groups in flexion and extension function, 2-PD, DASH, BCTQ, and VAS with respect to the affected fingers, and patients in both groups improved significantly before and after surgery. Treatment of only one wrist involved trimming to remove lesion-affected portions of tendon, which reappeared 1 year after surgery, and there was one case of poor recovery from greater piriformis muscle atrophy in both procedures. CONCLUSION: Regarding surgical treatment of patients with gouty carpal tunnel syndrome in which the gouty stone has invaded the superficial flexor tendons of the fingers, the diseased superficial flexor tendons can be selectively excised, and the postoperative mobility of the affected fingers may not be impaired.

Overexpression of Hsp104 by Causing Dissolution of the Prion Seeds Cures the Yeast [PSI+] Prion.

The yeast Sup35 protein misfolds into the infectious [PSI+] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [PSI+] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [PSI+] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.

Investigating the effect of prophylactic claw trimming on the interval between calving and first observed elevated locomotion score in pasture-based dairy cows.

AIMS: To evaluate, in a pasture-based dairy herd, the response to a three-time point hoof trimming regime on lameness incidence and time from calving to observation of an elevated locomotion score (LS). METHODS: This study was conducted on a 940-cow spring-calving herd in New Zealand's North Island between May 2018 and May 2019. Cows (n = 250) were randomly allocated to the hoof trimming group, with the remainder assigned to the non-trim cohort. One trained professional hoof trimmer used the five-step Dutch method to trim the hind feet of the trimming group. Throughout the subsequent production season, the whole herd was locomotion-scored fortnightly using the 4-point (0-3) Dairy NZ lameness score. Kaplan-Meier survival curves were used to assess the univariable effect of trimming on the interval between calving and first LS of ≥ 2 and first LS ≥ 1. A multivariable Cox proportional hazards regression was used to further evaluate the effect of trimming on time to elevated LS. RESULTS: Mean lameness (LS ≥ 2) prevalence was 2.6%, with 30% of cows having ≥ 4 observations during the study period when at least one LS was ≥ 2. For LS ≥ 1, mean prevalence was 40%, with 98.6% of cows having ≥ 4 observations during the study period when at least one LS was ≥ 1 during lactation. Hoof trimming had no apparent effect on the incidence of clinical lameness (LS ≥ 2) (trimmed vs. non-trimmed: 33.2% vs. 28.8%, respectively), but for LS ≥ 1, there was a small decrease in the incidence of LS ≥ 1 (trimmed vs. non-trimmed: 96.9% vs. 99.3%, respectively). The hazard of a cow having a first observed LS ≥ 2 in the control group was 0.87 (95% CI = 0.66-1.14) times that of the trimmed group; however, the hazard of a cow having a first LS ≥ 1 was 1.60 (95% CI = 1.37-1.88) times higher in the control than in the trimmed group. CONCLUSION AND CLINICAL RELEVANCE: On this farm, prophylactic hoof trimming had no clinically relevant impact on the incidence of clinical lameness and was not associated with clinically beneficial reductions in time to first observed LS ≥ 2. This may be because claw horn imbalance was not pronounced on this farm, with 53% of cows needing no trim on either hind limb on the first trimming occasion. Further research on the response to prophylactic trimming in pasture-based dairy cattle is required.

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein.

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.

Addressing Extreme Propensity Scores in Estimating Counterfactual Survival Functions via the Overlap Weights.

The inverse probability of treatment weighting (IPTW) approach is popular for evaluating causal effects in observational studies, but extreme propensity scores could bias the estimator and induce excessive variance. Recently, the overlap weighting approach has been proposed to alleviate this problem, which smoothly down-weights the subjects with extreme propensity scores. Although advantages of overlap weighting have been extensively demonstrated in literature with continuous and binary outcomes, research on its performance with time-to-event or survival outcomes is limited. In this article, we propose estimators that combine propensity score weighting and inverse probability of censoring weighting to estimate the counterfactual survival functions. These estimators are applicable to the general class of balancing weights, which includes IPTW, trimming, and overlap weighting as special cases. We conduct simulations to examine the empirical performance of these estimators with different propensity score weighting schemes in terms of bias, variance, and 95% confidence interval coverage, under various degrees of covariate overlap between treatment groups and censoring rates. We demonstrate that overlap weighting consistently outperforms IPTW and associated trimming methods in bias, variance, and coverage for time-to-event outcomes, and the advantages increase as the degree of covariate overlap between the treatment groups decreases.

Sensory deprivation leads to subpopulation-specific changes in layer 6 corticothalamic cells.

The effect of sensory deprivation on anatomical and physiological properties in two genetically defined types of layer 6 corticothalamic pyramidal cells in mouse somatosensory barrel cortex was investigated using in vitro electrophysiology. The two types analysed were the L6-Ntsr1 subtype, found preferentially in the upper region of layer 6 and projecting to both ventral posterior medial nucleus of the thalamus and posterior medial nucleus of the thalamus, and the L6-Drd1a subtype, located mostly in the lower regions of layer 6 and projecting to posterior medial nucleus. We found that the apical dendrite in L6-Ntsr1 cells is longer and more branched, compared with L6-Drd1a cells, and that the increase in firing frequency with increasing current stimulation is steeper in L6-Drd1a cells. Sensory deprivation was achieved clipping one row of whiskers from birth until the day of experiment (16 ± 2 days). Mice of this age are actively exploring. In L6-Ntsr1, but not in L6-Drd1a cells, sensory deprivation decreased apical and basal dendrite outgrowth, and calcium influx evoked by backpropagating action potentials. These results contribute to the ongoing functional characterisation of corticothalamic layer 6 cells and indicate differences in the postnatal cortical refinement of two distinct corticothalamic circuits.