PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends.

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions.

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

The Everted Acetabular Labrum: Patho-anatomy, Magnetic Resonance Imaging and Arthroscopic Findings of a Native Variant.

PURPOSE: The purpose of this study was to introduce a native labral variant, the everted acetabular labrum, and to describe the patho-anatomy, magnetic resonance imaging and magnetic resonance arthrogram (MRI/MRA) characteristics and the arthroscopic findings in this condition. METHODS: All primary hip arthroscopy procedures performed by the senior author between June 2013 and January 2020 were reviewed retrospectively. An everted acetabular labrum was identified as a segment of labrum that lacked apposition to the femoral head with the hip off traction. All everted labra were treated with labral advancement and repair with or without augmentation or reconstruction. The labrum-to-femoral head distance was measured in 3T MRI/MRA at the 1-2 o'clock position. A random selection of 38 hips without an everted labrum served as controls to compare radiographic parameters. RESULTS: A total of 68 hips were identified as having an everted labrum during the study period (mean age, 29.1 years), and 55 hips had advanced imaging available for review. MRI/MRA scans revealed the everted labrum to have a triangular shape in 17 hips (31%) and a blunted/round shape in 38 hips (69%), which differed significantly from controls (triangular 25/38 [66%], blunted 13/38 [34%], P < 0.001). The average labrum-to-femoral head distance was 1.4 mm for everted labra versus 0.0 mm for controls (P < 0.0001) and the mean labral lengths and widths were significantly shorter than those of controls (both P < 0.01). Of the hips, 8 underwent labral reconstruction or augmentation, and 61 underwent labral advancement/repair. CONCLUSION: The everted acetabular labrum is a native variant that is identifiable during hip arthroscopy by assessing the labral seal off traction. Preoperative MRI/MRA findings can be highly predictive of an everted labrum. Surgical treatment includes labral advancement and repair or reconstruction to restore contact between the labrum and the femoral head. LEVEL OF EVIDENCE: III, retrospective comparative study.

Phosphoproteomic Analysis of Subcutaneous and Omental Adipose Tissue Reveals Increased Lipid Turnover in Dairy Cows Supplemented with Conjugated Linoleic Acid.

Phosphoproteomics is a cutting-edge technique that can be utilized to explore adipose tissue (AT) metabolism by quantifying the repertoire of phospho-peptides (PP) in AT. Dairy cows were supplemented with conjugated linoleic acid (CLA, n = 5) or a control diet (CON, n = 5) from 63 d prepartum to 63 d postpartum; cows were slaughtered at 63 d postpartum and AT was collected. We performed a quantitative phosphoproteomics analysis of subcutaneous (SC) and omental (OM) AT using nanoUPLC-MS/MS and examined the effects of CLA supplementation on the change in the phosphoproteome. A total of 5919 PP were detected in AT, and the abundance of 854 (14.4%) were differential between CON and CLA AT (p ≤ 0.05 and fold change ± 1.5). The abundance of 470 PP (7.9%) differed between OM and SC AT, and the interaction treatment vs. AT depot was significant for 205 PP (3.5% of total PP). The integrated phosphoproteome demonstrated the up- and downregulation of PP from proteins related to lipolysis and lipogenesis, and phosphorylation events in multiple pathways, including the regulation of lipolysis in adipocytes, mTOR signaling, insulin signaling, AMPK signaling, and glycolysis. The differential regulation of phosphosite on a serine residue (S777) of fatty acid synthase (FASN) in AT of CLA-supplemented cows was related to lipogenesis and with more phosphorylation sites compared to acetyl-coenzyme A synthetase (ACSS2). Increased protein phosphorylation was seen in acetyl-CoA carboxylase 1 (ACACA;8 PP), FASN (9 PP), hormone sensitive lipase (LIPE;6 PP), perilipin (PLIN;3 PP), and diacylglycerol lipase alpha (DAGLA;1 PP) in CLA vs. CON AT. The relative gene expression in the SC and OM AT revealed an increase in LIPE and FASN in CLA compared to CON AT. In addition, the expression of DAGLA, which is a lipid metabolism enzyme related to the endocannabinoid system, was 1.6-fold higher in CLA vs. CON AT, and the expression of the cannabinoid receptor CNR1 was reduced in CLA vs. CON AT. Immunoblots of SC and OM AT showed an increased abundance of FASN and a lower abundance of CB1 in CLA vs. CON. This study presents a complete map of the SC and the OM AT phosphoproteome in dairy cows following CLA supplementation and discloses many unknown phosphorylation sites, suggestive of increased lipid turnover in AT, for further functional investigation.

Hydrogen-bonded mesomorphic complexes combining hydrophilic and fluorophilic molecular segments.

A 2,4-diamino-6-phenyl-1,3,5-triazine carrying a single oligo(ethylene oxide) (EO) chain has been investigated in binary mixtures with two-chain and three-chain semiperfluorinated benzoic acids. Mixtures of the triazine with three equivalents of the complementary acids exhibit a hexagonal columnar (Colh) mesophase. Docking of three acid molecules to the diaminotriazine nucleus leads to the formation of disc-like aggregates with a central hydrogen-bonded polar core surrounded by the peripheral fluoroalkyl chains, which self-assemble in columns arranged on a hexagonal lattice. The polar EO chains at the triazine cores do not segregate into a distinct sub-space but are included into the polar cores, providing increased flexibility of the cores with respect to conformations and available hydrogen bonding sites. In equimolar (1 : 1) triazine/benzoic acid mixtures macroscopic phase separation of excess triazine from the columnar LC phases occurs. These Colh phases are formed by [1 : 3] or [2 : 4] complexes, depending on the number of fluorinated chains. However, some of the excess triazine component is accommodated within the polar column cores, contributing to space filling and providing additional hydrogen bonding along the columns. Thus the EO chain, despite of being fixed at the periphery, assemble in the core region of the aggregates, this is distinct from the self-assembly of related alkyl or semiperfluoroalkyl substituted diaminotriazines with fluorinated benzoic acids, forming discrete disc-like [1 : 3] or rod-like [1 : 1] complexes. Obviously, the flexible EO chains decrease the effect of the aggregate shape on self-assembly and lead to an increased contribution of general amphiphilicity to self-assembly.

Determining glenoid component version after total shoulder arthroplasty.

BACKGROUND: Glenoid component loosening after total shoulder arthroplasty (TSA) may occur if retroversion is not corrected to <10°. However, accurately measuring postoperative glenoid component version has been difficult without postoperative computed tomography (CT), adding cost and radiation exposure outside of the standard radiographic follow-up. We present a new method to assess glenoid component version after TSA using only routine preoperative CT and postoperative radiographs (x-rays). METHODS: Preoperative glenoid version was measured using established methods with an axillary x-ray, 2-dimensional CT, and Glenosys software (Imascap, Plouzané, France). Postoperative glenoid component version and inclination were measured for 61 TSA patients using Mimics software (Materialise, Leuven, Belgium) with preoperative CT and postoperative x-rays. Four patients also had postoperative CTs. Glenoid implantation and imaging were performed on 14 cadavers, allowing validation of results against the gold standard postoperative CT glenoid retroversion measurement. RESULTS: Compared with the gold standard, retroversion and inclination measurement error was 2° ± 1° and 2° ± 1°, respectively. Average postoperative version correction was 6° ± 7°, with 35 of 61 patients (57%) corrected to <10° of retroversion. Correlation between preoperative version measurement methods was good to very good, except on the axillary x-ray. Patients not corrected to <10° of retroversion had significantly higher preoperative retroversion (14° ± 6°) than those corrected to <10° (6° ± 7°; P < .00001). CONCLUSIONS: Glenoid component retroversion after TSA can be accurately measured with a method using only routine preoperative CT and postoperative x-rays, validated to within 1.9° of the gold standard postoperative CT measurement. Future studies using this method may correlate glenoid retroversion correction with glenoid component longevity to help optimize shoulder arthroplasty outcomes.

Trends in Management and Complications of Anterior Cruciate Ligament Injuries in Pediatric Patients: A Survey of the PRiSM Society.

BACKGROUND: Anterior cruciate ligament (ACL) injuries have been recognized and treated with increasing frequency in children and adolescents. ACL reconstruction (ACLR) in skeletally immature patients has unique considerations and there is a large practice variation in the management of these injuries. The purpose of this study was to survey the members of the Pediatric Research in Sports Medicine (PRiSM) Society regarding their experience with the management and complications of pediatric ACL injuries. METHODS: A 15-question survey was distributed to 71 orthopaedic members of the Pediatric Research in Sports Medicine study group. RESULTS: Regarding treatment of an 8-year-old child with a complete ACL tear, 53% of respondents recommended iliotibial band reconstruction, 33% recommended all-epiphyseal reconstruction, and only 3% would treat nonoperatively in a brace. In adolescent patients with 2 years of growth remaining, 47% of respondents recommended a physeal "respecting" technique, 31% recommended a physeal-sparing technique, and 19% would perform an adult-style ACLR. There were 29 new cases of growth arrest reported. CONCLUSIONS: This study demonstrates that there have been major changes in the trends in pediatric ACLR over the past 15 years, and the most treatment variability is found among adolescent patients with about 2 years of growth remaining. The study also demonstrates that while the overall incidence of growth disturbance after ACLR remains low, new cases of growth disturbance continue to be identified. LEVEL OF EVIDENCE: Level V-survey of expert opinion and experience.

The effect of environmental temperature on reptilian peripheral blood B cell functions.

Recent studies have identified phagocytic B cells in a variety of species, yet little is understood about their function and how it is influenced by natural environmental variation, such as temperature. Phagocytic B-cells are present in red-eared slider turtles, Trachemys scripta, and the wide range of temperatures experienced by these ectotherms may have an effect on immunity, including B cell antibody secretion and phagocytosis. We examined the impact of environmental temperature on B cell function in vitro using phagocytic and ELISpot assays conducted at biologically relevant temperatures. We found a significant effect of temperature on antibody secretion, with maximal antibody secretion occurring at intermediate temperatures (estimated maximum of 28.8°C). There was no effect of temperature on phagocytosis. We also noted a difference in the efficiency of phagocytosis in this assay between B cells and non-B cells. Interestingly, in our in vitro assay, phagocytic B cells engulfed more foreign fluorescent beads per cell than phagocytes lacking surface immunoglobulin. This work sheds light on our understanding of phagocytic B cells and the importance of environmental temperature on the behavior of reptilian immune cells, which may have relevance for organismal fitness.

Polyphilic hydrogen bonded block molecules involving semiperfluorinated and silylated molecular fragments.

2,4-Diamino-6-phenyl-1,3,5-triazines grafted with one or two semiperfluorinated chains at the phenyl substituent have been investigated in binary mixtures with one-chain and two-chain oligo(dimethylsiloxy) terminated 4-alkoxy- and 3,4-dialkoxybenzoic acids. Equimolar mixtures of the triazines with the complementary acids form discrete double hydrogen-bonded heterodimers with an elongated central core. Mesomorphic properties are observed only if at least two terminal fluoroalkyl chains are attached to the triazine entity. Three to four heterodimers generate aggregates with preferably parallel adjusted rod-like H-bonded cores. The space requirement of the segregated peripheral fluoroalkyl and oligosiloxane segments considerably exceeds that of the central core region, leading to an overall circular geometry of the associates which organize in columnar LC phases. The chains initiate a local segregation which is however not long range and retains a time and space averaged two-dimensional hexagonal lattice.

Effect of Low Energy Waves on the Accumulation and Transport of Fecal Indicator Bacteria in Sand and Pore Water at Freshwater Beaches.

Elevated fecal indicator bacteria (FIB) in beach sand and pore water represent an important nonpoint source of contamination to surface waters. This study examines the physical processes governing the accumulation and distribution of FIB in a beach aquifer. Field data indicate E. coli and enterococci can be transported 1 and 2 m, respectively, below the water table. Data were used to calibrate a numerical model whereby FIB are delivered to a beach aquifer by wave-induced infiltration across the beach face. Simulations indicate FIB rapidly accumulate in a beach aquifer with FIB primarily associated with sand rather than freely residing in the pore water. Simulated transport of E. coli in a beach aquifer is complex and does not correlate with conservative tracer transport. Beaches with higher wave-induced infiltration rate and vertical infiltration velocity (i.e., beaches with higher beach slope and wave height, and lower terrestrial groundwater discharge) had greater E. coli accumulation and E. coli was transported deeper below the beach face. For certain beach conditions, the amount of FIB accumulated in sand over 5-6 days was found to be sufficient to trigger a beach advisory if eroded to surface water.

Chirality Synchronization of Hydrogen-Bonded Complexes of Achiral N-Heterocycles.

2,4-Diamino-6-phenyl-1,3,5-triazines carrying a single oligo(ethylene oxide) (EO) chain form an optically isotropic mesophase composed of a conglomerate of macroscopic chiral domains with opposite sense of chirality even though the constituent molecules are achiral. This mesophase was proposed to result from the helical packing of hydrogen-bonded triazine aggregates, providing long-range chirality synchronization. The results provide first evidence for macroscopic achiral symmetry breaking upon conglomerate formation in an amorphous isotropic phase formed by hydrogen-bonded associates of simple N-heterocycles that are related to prebiotic molecules.

Release of Escherichia coli from Foreshore Sand and Pore Water during Intensified Wave Conditions at a Recreational Beach.

Foreshore beach sands and pore water may act as a reservoir and nonpoint source of fecal indicator bacteria (FIB) to surface waters. This paper presents data collected at a fine sand beach on Lake Huron, Canada over three field events. The data show that foreshore sand erosion as wave height increases results in elevated Escherichia coli concentrations in surface water, as well as depletion of E. coli from the foreshore sand and pore water. E. coli initially attached to foreshore sand rather than initially residing in the pore water was found to be the main contributor to elevated surface water concentrations. Surface water E. coli concentrations were a function of not only wave height (and associated sand erosion) but also the time elapsed since a preceding period of high wave intensity. This finding is important for statistical regression models used to predict beach advisories. While calculations suggest that foreshore sand erosion may be the dominant mechanism for releasing E. coli to surface water during intensified wave conditions at a fine sand beach, comparative characterization of the E. coli distribution at a coarse sand-cobble beach suggests that interstitial pore water flow and discharge may be more important for coarser sand beaches.

Genetic and environmental variation in condition, cutaneous immunity, and haematocrit in house wrens.

BACKGROUND: Life-history studies of wild bird populations often focus on the relationship between an individual's condition and its capacity to mount an immune response, as measured by a commonly-employed assay of cutaneous immunity, the PHA skin test. In addition, haematocrit, the packed cell volume in relation to total blood volume, is often measured as an indicator of physiological performance. A multi-year study of a wild population of house wrens has recently revealed that those exhibiting the highest condition and strongest PHA responses as nestlings are most likely to be recruited to the breeding population and to breed through two years of age; in contrast, intermediate haematocrit values result in the highest recruitment to the population. Selection theory would predict, therefore, that most of the underlying genetic variation in these traits should be exhausted resulting in low heritability, although such traits may also exhibit low heritability because of increased residual variance. Here, we examine the genetic and environmental variation in condition, cutaneous immunity, and haematocrit using an animal model based on a pedigree of approximately 2,800 house wrens. RESULTS: Environmental effects played a paramount role in shaping the expression of the fitness-related traits measured in this wild population, but two of them, condition and haematocrit, retained significant heritable variation. Condition was also positively correlated with both the PHA response and haematocrit, but in the absence of any significant genetic correlations, it appears that this covariance arises through parallel effects of the environment acting on this suite of traits. CONCLUSIONS: The maintenance of genetic variation in different measures of condition appears to be a pervasive feature of wild bird populations, in contradiction of conventional selection theory. A major challenge in future studies will be to explain how such variation persists in the face of the directional selection acting on condition in house wrens and other species.

Neonatal body condition, immune responsiveness, and hematocrit predict longevity in a wild bird population.

Measures of body condition, immune function, and hematological health are widely used in ecological studies of vertebrate populations, predicated on the assumption that these traits are linked to fitness. However, compelling evidence that these traits actually predict long-term survival and reproductive success among individuals in the wild is lacking. Here, we show that body condition (i.e., size-adjusted body mass) and cutaneous immune responsiveness to phytohaemagglutinin (PHA) injection among neonates positively predict recruitment and subsequent longevity in a wild, migratory population of house wrens (Troglodytes aedon). However, neonates with intermediate hematocrit had the highest recruitment and longevity. Neonates with the highest PHA responsiveness and intermediate hematocrit prior to independence eventually produced the most offspring during their lifetime breeding on the study site. Importantly, the effects of PHA responsiveness and hematocrit were revealed while controlling for variation in body condition, sex, and environmental variation. Thus, our data demonstrate that body condition, cutaneous immune responsiveness, and hematocrit as a neonate are associated with individual fitness. Although hematocrit's effect is more complex than traditionally thought, our results suggest a previously underappreciated role for this trait in influencing survival in the wild.

Superior labrum anterior-to-posterior repair incidence: a longitudinal investigation of community and academic databases.

BACKGROUND: Superior labrum anterior-to-posterior (SLAP) lesion repair is controversial regarding indications and potential complications. METHODS: Databases were used to determine the SLAP repair incidence compared with all orthopaedic procedures over a period of 10 years. In part A, the New York Statewide Planning and Research Cooperative System ambulatory surgery database was investigated from 2002 to 2009. In part B, the California Office of Statewide Health Planning and Development ambulatory surgery database was investigated from 2005 to 2009. In part C, the American Board of Orthopaedic Surgery (ABOS) database was investigated from 2003 to 2010. RESULTS: In part A, from 2002 to 2009, there was a 238% increase in SLAP repair volume compared with a 125% increase in all orthopaedic procedures. In part B, from 2005 to 2009, there was a 20.17% increase in SLAP repair volume compared with a decrease of 13.64% in all orthopaedic procedures. In part C, among candidates performing at least 1 SLAP repair, there was no statistically significant difference in likelihood of performing a SLAP repair (95% confidence interval, 0.973-1.003) in 2010 as compared with 2003 (P > .10). CONCLUSIONS: There has been a significant increase in the incidence of SLAP repairs in the past 10 years in statewide databases. This pattern was not seen in the ABOS database, in which the annual volume of SLAP repairs remained stable over the same period. This suggests that SLAP lesions have been over-treated with surgical repair but that part II ABOS candidates are becoming more aware of the need to narrow indications. LEVEL OF EVIDENCE: Epidemiology study, database analysis.

The Everted Acetabular Labrum: Outcomes of Surgical Management.

BACKGROUND: An everted acetabular labrum (EL) is a pathologic variant in which the labrum is flipped to the capsular side of the acetabular rim. An iatrogenic EL is a known complication of a poorly executed labral repair, and a recent study described the native acetabular EL. PURPOSE: To analyze surgical outcomes after advancement or reconstruction of an EL in a native hip. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: This was a multicenter retrospective review of prospectively collected data on primary hip arthroscopic surgeries performed between 2013 and 2023. An EL was identified arthroscopically as a labrum-femoral head gap while off traction in the native hip. All patients with EL who were analyzed in this study underwent arthroscopic labral repair and advancement or labral augmentation or reconstruction. Patients with hip dysplasia also underwent periacetabular osteotomy with or without a derotational femoral osteotomy. Patient-reported outcomes (PROs) were assessed using the 12-item International Hip Outcome Tool (iHOT-12) and the Nonarthritic Hip Score. PROs were obtained preoperatively and up to 24 months after surgery. PROs were compared with those of a case-matched control cohort in a 1:2 ratio. Only patients with PROs available at ≥1 year postoperatively were included in the outcome analysis. RESULTS: A total of 111 patients (129 hips) with EL during the study period were identified, with PROs available in 96 hips. The mean age of patients with EL was 30.5 years, and women made up 87% of the cohort. Of the 129 hips with an EL, an isolated diagnosis of an EL was present in 11.6% of hips. Deficient acetabular coverage (lateral center-edge angle <25°) was seen in 40.6% of EL hips. No difference was seen in iHOT-12 scores between EL and control groups at 12- or 24-month follow-up (P = .18 and .94, respectively). Patients with EL reported a significant improvement of PROs at latest follow-up (P < .001 for iHOT-12 and Nonarthritic Hip Score). CONCLUSION: Surgical management of a native EL with restoration of the labral seal on the femoral head and correction of concomitant pathologies resulted in significant clinical improvement, with postoperative outcome scores comparable to those of patients without an EL. These findings provide evidence supporting surgical intervention for a native EL.

Solutes unmask differences in clustering versus phase separation of FET proteins.

Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride. These differences on the nanoscale, gleaned using a suite of methods deployed across a wide range of protein concentrations, are prevalent and can be unmasked even though the driving forces for phase separation remain unchanged in glutamate versus chloride. Strikingly, differences in anion-mediated interactions that drive clustering saturate on the micron-scale. Beyond this length scale the system separates into coexisting phases. Overall, we find that sequence-encoded interactions, mediated by solution components, make synergistic and distinct contributions to the formation of pre-percolation clusters in sub-saturated solutions, and to the driving forces for phase separation.

Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle.

Aging is typically associated with a decrease in immune function. However, aging does not affect each branch of the immune system equally. Because of these varying effects of age on immune responses, aging could affect taxa differently based on how the particular taxon employs its resources towards different components of immune defense. An example of this is found in the humoral immune system. Specific responses tend to decrease with age while non-specific, natural antibody responses increase with age. Compared with mammals, reptiles of all ages have a slower and less robust humoral immune system. Therefore, they may invest more in non-specific responses and thus avoid the negative consequences of age on the immune system. We examined how the humoral immune system of reptiles is affected by aging and investigated the roles of non-specific, natural antibody responses and specific responses by examining several characteristics of antibodies against lipopolysaccharide (LPS) in the red-eared slider turtle. We found very little evidence of immunosenescence in the humoral immune system of the red-eared slider turtle, Trachemys scripta, which supports the idea that non-specific, natural antibody responses are an important line of defense in reptiles. Overall, this demonstrates that a taxon's immune strategy can influence how the immune system is affected by age.

Case report: Teat stenosis in a suckler cow.

Bovine veterinarians are regularly confronted with teat lesions in cows. The number of studies on the diagnosis and treatment of teat lesions as well as the exchange of practical experience among clinicians are extensive in dairy cows compared with suckler cows. The aim of this case report was to describe the successful treatment of teat stenosis in a suckler cow and discuss possible challenges. A four-year-old Simmental cow, in her third lactation and 4 days in milk, was referred to our clinic along with her calf because of teat stenosis in the front left quarter. The owner had repeatedly used a rigid teat cannula in an attempt to relieve the stenosis during the previous lactation. However, the cow had refused to allow the current calf to suckle the affected teat and resisted attempts by the owner to cannulate the teat. The results of clinical examination, ultrasonography, and milk sampling showed stenosis of the proximal, middle, and distal parts of the front left teat cistern, accompanied by thelitis and cisternitis and mild chronic clinical mastitis. Based on published recommendations, treatment of the thelitis, cisternitis, and mastitis was initiated before resolution of the stenosis surgically. The first week of treatment included the administration of an intramammary product containing cefapirin and prednisolone, a systemic non-steroidal anti-inflammatory drug, a wax teat-boogie, and bandaging of the teat. Thereafter, the treatment was reduced to insertion of a wax-teat boogie and bandaging. Conservative treatment resulted in resolution of the mastitis, cisternitis, and stenosis in the proximal and middle parts of the teat, which had most likely been caused by repeated cannulation of the teat by the owner. Lateral theloscopy was then used to remove the distal stenosis, which was the primary lesion. Healing of the surgical wound and resolution of the swelling occurred several days postoperatively, and the calf's first attempt to suckle the teat was successful. The cow and calf were discharged from the clinic 2 weeks after surgery. A follow-up visit 4.5 months after surgery revealed that the calf was still nursing the teat and the operated quarter was producing a normal amount of milk.

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions.

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.