Effects of feeding an inoculated corn silage with or without a direct-fed microbial on dry matter intake, milk production, and nutrient digestibility of high-producing lactating Holstein cows.

This study evaluated the effects of inoculating corn silage and/or feeding a direct-fed microbial (PRO) on performance and nutrient digestibility of lactating dairy cows. At harvesting, corn silage was treated either with water (culated or not [CON]) or Lactococcus lactis and Lentilactobacillus buchneri (INC; SiloSolve FC) at 1.5 × 105 cfu/g of corn silage. Ten mini silos and one farm-scale silo bunker per treatment were prepared for the laboratory and the lactating dairy cow trial, respectively. Five mini silos per treatment were opened on days 2 or 90 post-ensiling for pH measurement, as well as chemical analysis and aerobic stability, respectively. The farm-scale silo bunkers were opened 77 d post-ensiling for the beginning of the lactating cow trial. Eighty lactating Holstein cows were assigned in a 2 × 2 factorial design to: (1) CON silage without PRO (CON-CON; n = 20), (2) CON silage with PRO at 14 g/head/d (CON-PRO; n = 20), (3) INC silage without PRO (INC-CON; n = 20), and (4) INC silage with PRO at 14 g/head/d (INC-PRO; n = 20). Concurrently with the feeding trial, eight cows per treatment were chosen for nutrient digestibility. The pH of the corn silage was not affected by the silage inoculant (P ≥ 0.29), but INC yielded greater concentration of acetic acid and longer aerobic stability (P < 0.01). Dairy cows fed INC had a lower mean total dry matter intake (DMI), milk protein content, and somatic cell counts vs. CON (P ≤ 0.02). On the other hand, milk and fat- and protein-corrected milk (FPCM) production efficiency, milk urea-N, DM, crude protein, and starch digestibility were greater for INC-fed cows (P ≤ 0.03). Feeding direct-fed microbials (DFM) improved mean body weight, milk yield, and FPCM, as well as milk protein and lactose yield (P ≤ 0.05), but reduced milk fat and protein content (P = 0.02). A silage inoculant × DFM interaction was observed for milk production efficiency, milk protein and lactose content, and somatic cell count (P ≤ 0.05). Dairy cows fed INC-CON had a greater milk production efficiency and milk lactose content (P ≤ 0.04), but INC-PRO had lower milk protein content and SCC (P ≤ 0.03). In summary, inoculating L. lactis and L. buchneri increased acetic acid content and aerobic stability of corn silage, reduced DMI, but improved milk production efficiency and nutrient digestibility of lactating Holstein dairy cows. On the other hand, feeding PRO improved milk, protein, and lactose yield. Additionally, combining the feeding of an inoculated corn silage with PRO reduced milk somatic cell count.

The Effect of Probiotics in a Milk Replacer on Leukocyte Differential Counts, Phenotype, and Function in Neonatal Dairy Calves.

Probiotics have been investigated for many health benefits; however, few studies have been performed to determine the effects of oral probiotics on peripheral blood and respiratory immune cells in cattle. Our objectives were to determine changes in health and growth status, differential blood cell counts and function, and blood and lung cell function using flow cytometry and PCR in dairy calves fed a milk replacer with (PRO, N = 10) or without (CON, N = 10) the addition of probiotics to the milk replacer and dry rations from birth to weaning. Performance and clinical scores were not different between the treatment groups. Treatment-by-day interactions for peripheral blood leukocyte populations differed in cell number and percentages. A greater percentage of leukocytes expressed the cell surface markers CD3, CD4, CD8, CD11b, and CD205 on d 21 in CON animals. Lung lavages were performed on five animals from each treatment group on d 52. There were no differences between treatment groups for the expression of cytokines and Toll-Like Receptors as measured using Polymerase Chain Reaction, possibly due to the small sample size. Oral probiotics appear to affect peripheral blood immune cells and function. Their effect on overall calf health remains to be determined.

Evaluation of a Bacillus-based direct-fed microbial probiotic on in vitro rumen gas production and nutrient digestibility of different feedstuffs and total mixed rations.

We evaluated the effects of a Bacillus-based direct-fed microbial (DFM) on total in vitro gas production, dry matter (DM), neutral detergent fiber (NDF), and starch disappearance of different feedstuffs and total mixed rations (TMR) in three different experiments. In experiment 1, six single fiber-based feedstuffs were evaluated: alfalfa hay, buffalo grass, beet pulp, eragrostis hay, oat hay, and smutsvinger grass. Experimental treatments were control (with no probiotic inoculation; CON) or incubation of a probiotic mixture containing Bacillus licheniformis and B. subtilis (3.2 × 109 CFU/g; DFM). The calculation of DFM dose under in vitro conditions was based on the assumption of a rumen capacity of 70 liter and the dose of 3 g of the DFM mixture/head/d (9.6 × 109 CFU). Total in vitro gas production, DM, and NDF disappearance were evaluated at 24- and 48 h posttreatment incubation. Mean treatment effects were observed at 24- and 48 h gas production (P < 0.0001), as DFM incubation increased in vitro gas production by 5.0% and 6.5%, respectively. For nutrient digestibility, mean DM digestibility was increased at 48 h (P = 0.05), whereas mean NDF digestibility increased at both timepoints by incubating DFM in vitro (P ≤ 0.02). In experiment 2, nine commercial dairy TMR were collected and evaluated for the same variables and treatments described in experiment 1, with the additional analysis of starch digestibility at 7 h post in vitro incubation. The only difference was the concentration of the DFM included, being representative for a dosage of 8.8 × 109 CFU/head/d. In vitro gas production was increased only at 48 h due to DFM incubation (P = 0.05), whereas DM and NDF digestibility were improved at 24 and 48 h (P ≤ 0.02). No treatment effects were observed on in vitro starch digestibility (P = 0.31). In experiment 3, a combined analysis of DM and NDF digestibility was performed by using quality values (NDF and crude protein or CP) of 16 substrates. Regardless of CP and NDF levels of the substrates, DFM improved in vitro 24 and 48 h DM and NDF digestibility (P ≤ 0.03). In summary, incubating a Bacillus-based DFM (B. licheniformis and B. subtilis; BOVACILLUS) improved mean in vitro gas production, DM, and NDF digestibility of single feedstuffs and commercial dairy TMR, highlighting the potential of this combination of Bacillus spp. to improve nutrient utilization, mainly fiber.

Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves.

Introduction: Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. Recent data have also supported a gut-lung axis regarding the bacterial populations (microbiomes) of the two locations; however, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. We hypothesized that probiotic treatment would result in changes in the lung microbiome as measured in lung lavage fluid. Our overall goal was to characterize bacterial populations in the lungs of calves fed probiotics in milk replacer and dry rations from birth to weaning. Methods: A group of 20 dairy calves was split into two treatment groups: probiotic (TRT; N = 10, milk replacer +5 g/d probiotics; Bovamine Dairy, Chr. Hansen, Inc., Milwaukee, WI) and control (CON; N = 10, milk replacer only). On day 0, birth weight was obtained, and calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group and then included in their dry ration. Lung lavages were performed on day 52 on five random calves selected from each treatment group. DNA was extracted from lavage fluid, and 16S ribosomal RNA (rRNA) gene hypervariable regions 1-3 were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for the identification of the bacterial taxa present. Taxa were classified into both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). Results: Overall, the evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to the control calves were significantly different based on the OTU dataset (p < 0.05) and approached significance for the ASV dataset (p < 0.06). Additionally, when comparing the diversity of taxa in lung lavage samples to nasal and tonsil samples, taxa diversity of lung samples was significantly lower (p < 0.05). Discussion: In conclusion, analysis of the respiratory microbiome in lung lavage samples after probiotic treatment provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more than the gut microbiome.

Efficacy of Two Probiotic Products Fed Daily to Reduce Clostridium perfringens-Based Adverse Health and Performance Effects in Dairy Calves.

Clostridium perfringens is a spore-forming, anaerobic bacterium which produces toxins and exoenzymes that cause disease in calves, especially necro-hemorrhagic enteritis-associated diarrhea often resulting in death. Clostridium infections are currently being treated with antibiotics, but even with the prudent administration of antibiotics, there are significant rates of recurrence. Probiotics, an alternative to antibiotics, are commonly employed to prevent clostridial infections. The objectives of our study were to demonstrate that two commercially available products, when used as daily, direct-fed microbials, are effective in reducing adverse effects of an experimentally induced C. perfringens infection in dairy calves. We conducted a single site efficacy study with masking using a randomized design comprising 10 calves allocated to 3 treatment groups (probiotic 1, probiotic 2, and control). The procedures such as general health scores, body weight, blood samples, and fecal sample collections were done followed by experimental challenge of calves with C. perfringens. Daily feeding of L. animalis LA51 and P. freudenreichii PF24 without or with Bacillus lichenformis CH200 and Bacillus subtilis CH201, before, during and after an oral challenge of C. perfringens significantly reduced the incidence and severity of diarrhea while improving general impression and appearance scores of calves. Most notably, survival of calves in the two probiotic-fed groups was significantly higher than for control calves and further substantiates the potential economic and health benefits of feeding effective probiotics.

Efficacy of Lactobacillus animalis and Propionibacterium freudenreichii-Based Feed Additives in Reducing Salmonella-Associated Health and Performance Effects in Commercial Beef Calves.

Salmonella enterica, which causes typhoid fever, is one of the most prevalent food-borne pathogens. Salmonellosis in cattle can greatly impact a producer's income due to treatment costs, decreased productivity of the herd, and mortality due to disease. Current methods of treatment and prevention for salmonellosis consist of antibiotics and vaccinations, but neither of these options are perfect. Probiotics, categorized as antibiotic alternatives, are living microorganisms that are added to animal feeds in appropriate quantities in order to benefit health and productivity in adult and newborn livestock. The objective of this study was to demonstrate that Lactobacillus animalis and Propionibacterium freudenreichii, when used as a direct-fed microbial, was effective in reducing the adverse effects of experimentally induced Salmonella infection in beef calves. We conducted a single site efficacy study with masking using a randomized design comprising two groups of ten beef calves allocated to two treatment groups (control and probiotic). Procedures such as determining general health scores and body weight and collecting fecal samples were carried out following the experimental challenge of calves with Salmonella Typhimurium. The presence of at least one CFU of bacteria in feces was significantly higher among animals in the control than in the probiotic group, which was higher on days 0 to 7 than on days 8 to 14 (p = 0.012). Animals in the control group had a significantly higher presence of abnormal diarrhea scores than animals in the probiotic group (p < 0.001). Most notably, other health benefits in probiotic-fed group calves were obviously better than those for control calves and further substantiates the potential economic and health benefits of feeding effective probiotics.

Variation in the response of bovine alveolar lavage cells to diverse species of probiotic bacteria.

OBJECTIVE: Probiotics are fed to improve enteric health, and they may also affect respiratory immunity through their exposure to the upper respiratory tract upon ingestion. However, their effect on the respiratory system is not known. Our aim was to determine how probiotics affect functions and markers of bronchoalveolar lung lavage cells (BAL) isolated from lungs of calves at slaughter. RESULTS: Treatments consisted of ten probiotic species and one control treatment. Probiotics and BAL were incubated 1:1 for 2 h at 37 °C and 5% CO2. The cell surface markers measured included CD14, CD205, and CD18, and E. coli bioparticles were used to measure phagocytosis and oxidative burst. Differences were considered significant at P ≤ 0.05 and were noted for percent cells fluorescing and mean fluorescence intensity for CD14 and CD205. Additionally, oxidative burst was different as measured by both percentage of cells fluorescing and mean fluorescence intensity, and phagocytosis differed among species as measured by mean fluorescence intensity. Overall, probiotic species differed in their ability to suppress or increase leukocyte function showing that probiotic bacteria differentially modulate BAL.

Evaluation of Oxathiapiprolin for the Management of Sunflower Downy Mildew.

Downy mildew is a yield-limiting disease of sunflower, caused by the pathogen Plasmopara halstedii. Zoospore infection of root tissue shortly after planting results in systemic infection, causing postemergence damping off or severe stunting and head sterility. Although fungicide-applied seed treatments can be an effective management tool, the pathogen is resistant to phenylamide fungicides in many growing regions, and other available fungicides have limited efficacy. Oxathiapiprolin, the first member of the piperidinyl thiazole isoxazoline fungicides, was evaluated for efficacy on downy mildew in field trials conducted from 2011 to 2015 in North Dakota. Throughout the course of the study, the rate range was narrowed from active ingredient (a.i.) at 0.45 to 116.0 µg a.i. seed-1 to an optimal effective rate of 9.37 to 18.75 µg a.i. seed-1. Within that optimal range, the downy mildew incidence of sunflower planted with oxathiapiprolin-treated seed was significantly lower than the incidence in the nontreated sunflower in all 11 trials with disease pressure. Additionally, downy mildew incidence of sunflower planted with oxathiapiprolin-treated seed was significantly lower than sunflower planted with competitive commercially available fungicide-treated seed in 10 of those 11 trials. The use of oxathiapiprolin by sunflower growers is likely to reduce disease incidence and subsequent yield loss to downy mildew.

Care Transition Experiences of Older Veterans and Their Caregivers.

This study's main objective was to examine care transition experiences of older veterans and their caregivers. Fifty patients age 65 years and older, discharged from a Veterans Affairs Medical Center hospital, completed the Care Transitions Measure-15 survey three to four weeks postdischarge. Seven patients and six caregivers participated in semistructured interviews. Overall, the quality of care transitions was rated as good; however, some items were indicated as problematic for veterans. Themes that emerged included agreeableness, frustration with complex information, caregiver education, and the timing and methods of information delivery. These findings have implications for all clinical staff working with veterans, and particularly for social workers facilitating care transitions for veterans and their caregivers.

Development of a model for teaching manipulation of a distal radial fracture.

BACKGROUND: Recent reports indicate that there is a trend toward the use of models and simulators to teach basic orthopaedic operative skills. We describe a high-fidelity model for teaching basic orthopaedic fracture manipulation. METHODS: A model of a distal radial fracture was developed and validated by orthopaedic surgeons with different levels of experience, who examined and manipulated the model and then completed a structured questionnaire. RESULTS: The participants mostly agreed (designated as 4 in the questionnaire) that the model performed well on the degree to which the manipulation represented real life. The participants agreed (designated as 5 in the questionnaire) that the model scored high on the appearance and feel of the fracture and its ability to be used as a teaching tool. There was no difference between perceptions of participants with regard to different levels of occupational experience or model use. CONCLUSIONS: We believe that this model could be used to teach the skill of closed reduction of a distal radial fracture in the preclinical setting.

Harmonin inhibits presynaptic Cav1.3 Ca²âº channels in mouse inner hair cells.

Harmonin is a scaffolding protein that is required for normal mechanosensory function in hair cells. We found a presynaptic association of harmonin and Ca(v)1.3 Ca(2+) channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling.

At the presynaptic active zone, Ca²+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca²+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca²+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca²+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca²+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca²+ channels and vesicles, and (2) promote vesicle replenishment.

Allele-specific effects of human deafness gamma-actin mutations (DFNA20/26) on the actin/cofilin interaction.

Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients.

In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment.

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

Evaluation of rapidly progressive dementia.

The evaluation of patients with rapidly progressive dementia can be challenging, largely due to the rarity of the conditions responsible for such fulminant presentations of dementia. This article presents a framework for categorizing such disorders based on the underlying pathophysiology, including degenerative, inflammatory, vascular, toxic, metabolic, neoplastic, and infectious causes of rapidly progressive dementia. Diagnostic approaches, representative imaging abnormalities, and treatment options for these uncommon conditions are also presented.

Effects of human deafness gamma-actin mutations (DFNA20/26) on actin function.

Six point mutations in non-muscle gamma-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each gamma-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize.

Role of the N-terminal negative charges of actin in force generation and cross-bridge kinetics in reconstituted bovine cardiac muscle fibres.

Mutant yeast actins were used to determine the role of actin's N-terminal negative charges in force generation. The thin filament was selectively removed from bovine cardiac skinned muscle fibres by gelsolin, and the actin filament was reconstituted from purified G-actin. In this reconstitution, yeast wild-type actin (2Ac: two N-terminal negative charges), yeast mutant actins (3Ac and 4Ac), and rabbit skeletal muscle actin (MAc) were used. The effects of phosphate, ATP and ADP on force development were studied at 25 degrees C. With MAc, isometric tension was 77% of the initial tension owing to the lack of a regulatory system. With 2Ac, isometric tension was 10% of the initial tension; with 3Ac, isometric tension was 23%; and with 4Ac, isometric tension was 44%. Stiffness followed a similar pattern (2Ac < 3Ac < 4Ac < MAc). A similar trend was observed during rigor induction and relaxation. Sinusoidal analysis was performed to obtain the kinetic constants of the cross-bridge cycle. The results showed that the variability of the kinetic constants was < or = 2.5-fold among the 2Ac, 4Ac and MAc muscle models. When the cross-bridge distribution was examined, there was no significant reapportionment among these three models examined. These results indicate that force supported by each cross-bridge is modified by the N-terminal negative charges of actin, presumably via the actomyosin interface. We conclude that two N-terminal negative charges are not adequate, three negative charges are intermediate, and four negative charges are necessary to generate force.

Opioid contract use is associated with physician training level and practice specialty.

Opioid contracts are widely used to manage opioid prescribing in the treatment of pain conditions, but they are not well studied. A notable gap in our knowledge of opioid contracts involves the factors that determine their use. As an initial inquiry, this study evaluated the responses of a Web-based survey of trainees and faculty in an academic medical training context to determine correlates of opioid contract use. All paid faculty, third- and fourth-year medical students, and residents in The University of Oklahoma College of Medicine were invited via email to participate in a Web-based survey of their attitudes and prescribing practices related to controlled prescription drugs. Respondents composing a subgroup of those who replied to the survey were identified by their prescription of opioids and by their designation that pain was the most likely diagnosis for which they would prescribe a controlled drug. Chi-square analysis was used to determine any correlation between contract use and respondents' demographic variables and categorical survey responses. Analysis of variance was used to determine any correlation between contract use and survey responses that involved continuous variables. Our results showed that opioid contract use was significantly associated with resident status, primary care specialty, participant estimation of alcohol and illicit drug abuse by patients, and the participant's assessment of the risks in general of prescribing controlled drugs. A majority of contract users reported that the use of this tool increased their sense of mastery and comfort with prescribing controlled drugs. The factors associated with opioid contract use found in this study suggest there are significant prescriber-specific determinants of the use of the tool, including training level, medical specialty, and risk appraisals. Opioid contracts' effects on mastery and comfort of the physician with prescribing opioids suggest that they may play an important role in facilitating appropriate pain management with opioids. Further study is needed to elucidate environmental and patient-specific factors that may influence opioid contract use.

An intermediate form of ADP-F-actin.

With yeast actin, contrary to other actins, filament formation, ATP hydrolysis, and Pi release are concurrent at low actin concentrations, the condition usually employed to assess actin polymerization. This observation leads to a question concerning the conformation of the filament barbed end that might be recognized by specific actin-binding proteins. To try to detect possible new actin polymer conformations that might be intermediate in the pathway leading to mature F-actin, we monitored the change in intrinsic tryptophan fluorescence of yeast and muscle actins polymerized at pH 6 to accelerate the rate of filament formation. This allowed temporal resolution of the Pi release process from the slower process of polymerization. With both actins, we detected a biphasic instead of the usual monophasic fluorescence change, a rapid decrease that tracks with filament formation followed by a slower rebound (the second phase). This second phase postpolymerization conformational change requires Pi release and occurs nearly coincident with its release. The addition of Pi causes this second phase response to disappear, and the inclusion of Pi during polymerization prevents its appearance. At pH 7.5, with higher yeast actin concentrations to accelerate polymerization, a two-phase fluorescence change is also observed. In this case, the second phase change lags substantially behind Pi release. Pi release could also be resolved from polymer formation. V159N yeast actin, hypothesized previously as remaining in a postpolymerization ATP-like state, exhibits the same two-phase intrinsic tryptophan fluorescence behavior as wild-type yeast actin. Together, these observations demonstrate the presence of an intermediate filament state between ADP-Pi and mature ADP-F-actin.