SMT-738: a novel small-molecule inhibitor of bacterial lipoprotein transport targeting Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CREs) are described by the Centers for Disease Control as an urgent threat, and there is a critical need for new therapeutic agents able to treat infections caused by these pathogens. Herein, we describe the microbiological profile, the mechanism f action, and the in vitro safety as well as the pharmacokinetic (PK)/PD profile of SMT-738, a small molecule belonging to a new chemical class. SMT-738 is active against Enterobacterales [including multi-drug-resistant Escherichia coli with 90% of isolates having a minimum inhibitory concentration (MIC90) of 1 µg/mL and Klebsiella pneumoniae 2 µg/mL] and inactive against a broad panel of Gram-negative and Gram-positive pathogens. SMT-738 displays rapid bactericidal activity (2-4 h) and has a low propensity for resistance development (less than ~10-9). Characterization of resistant mutants following exposure to SMT-738 identified mutations within the lipoprotein transport complex (LolCDE), a clinically unexploited and essential bacterial molecular target in Gram-negative bacteria. SMT-738 has a promising in vitro toxicology profile. Furthermore, PK studies demonstrated that when dosed intravenously, SMT-738 maintained exposure levels across infection sites (bloodstream/urinary tract/lung). Proof-of-concept studies across multiple murine in vivo infection models (bloodstream/pneumonia/urinary tract) demonstrated that SMT-738 significantly reduced the bacterial burden compared to baseline and vehicle control. SMT-738 represents a promising novel drug candidate being developed to address clinically challenging serious life-threatening infections caused by highly resistant Enterobacteriaceae including CRE.

Early prediction of respiratory disease in preweaning dairy calves using feeding and activity behaviors.

Bovine respiratory disease (BRD) represents one of the major disease challenges affecting preweaning dairy-bred calves. Previous studies have shown that differences in feeding and activity behaviors exist between healthy and diseased calves affected by BRD. The aim of this study was to develop and assess the accuracy of models designed to predict BRD from feeding and activity behaviors. Feeding and activity behaviors were recorded for 100 male preweaning calves between ~8 to 42 d of age. Calves were group housed with ad libitum access to milk via automatic milk feeders, water, starter diet, and straw. Activity was monitored via a leg-mounted accelerometer. Health status of individual calves was monitored daily using an adapted version of the Wisconsin Scoring System to identify BRD. Three models were created to predict disease: (1) deviation from normal lying time based on moving averages (MA); (2) random forest (RF), a machine learning technique based on feeding and activity variables; and (3) a combination of RF and MA output. For the MA model, lying time was predicted based on behavior over previous days (3- and 7-d MA) and the expected value for the current day (based on calf age; measured using accelerometers). Data were not split into training and test data sets. Occasions when the actual lying time increased >9% of predicted lying time were classified as a deviation from normal and a disease alert was provided. Both feeding and activity behaviors were included within the RF model. Data were split into training (70%) and test (30%) data sets based on disease events. Events were classified as 2 d before, the day(s) of the disease event, and 2 d after the event. Accuracy of models was assessed using sensitivity, specificity, balanced accuracy, and Matthews correlation coefficient (MCC). If a positive disease prediction agreed with an actual disease event within a 3-d rolling window, it was classified as a true positive. Stand-alone models (RF; MA) showed high specificity (0.95; 0.97), moderate sensitivity (0.35; 0.43), balanced accuracy (0.65; 0.64), and MCC (0.25; 0.29). Combining outputs increased accuracy (specificity = 0.95, sensitivity = 0.54, balanced accuracy = 0.75, MCC = 0.36). The work presented is the first to demonstrate the use of modeling data derived from precision livestock farming techniques that monitor feeding and activity behaviors for early detection of BRD in preweaning calves, offering a significant advance in health management of youngstock.

A prospective cohort study of digital cushion and corium thickness. Part 2: Does thinning of the digital cushion and corium lead to lameness and claw horn disruption lesions?

The aim of this study was to determine whether a decrease in thickness of the sole soft tissues (SST) beneath the flexor tuberosity of the distal phalanx (i.e., the digital cushion and corium) predisposed a claw to develop claw horn disruption lesions (CHDL) or a leg to lameness. Data were analyzed from a longitudinal study of 179 cows, which had been examined at 5 assessment points -8, +1, +9, +17, and +29 wk relative to their first, second, third, or fourth calving. At each assessment point, SST were measured using ultrasonography. Additional assessment point data included sole lesions and back fat thickness (BFT), and cows had been locomotion scored every 2 wk from calving. One hundred fifty-eight cows completed the study. Separate logistic regression survival analyses were constructed to assess the outcomes, either lameness on a leg or CHDL on a claw; combinations of lameness and lesions were tested as outcomes. Cow level variables tested included farm and lactation number. Variables were tested describing previous SST thickness, minimum previous SST thickness, BFT, and change in either variable between prior assessment points. Prior lesions/lameness strongly predicted repeat cases and the final models had the outcome first lesion or lameness on a claw or leg. In the reported lameness models, lameness was defined as a leg being recorded as lame twice within 3 consecutive scores, and in the reported lesion models, lesion was defined as the first presence of either a sole ulcer or a severe sole hemorrhage on a claw. Thin SST increased the likelihood of lesion occurrence; thin SST on the lateral claw predicted subsequent lameness on a leg. Thin BFT and thinning of BFT between previous assessment points increased the likelihood of future lesion occurrence. Thin SST and thinning of BFT had additional effects on the likelihood of lesion occurrence, suggesting that BFT and sole SST had independent effects on lesion occurrence. However, change in SST thickness between assessment points did not influence the likelihood of future lesions or lameness. This suggests that thin SST were not simply a result of depletion of body fat and challenges the theory that thinning of the digital cushion with body fat mobilization leads to CHDL. Other possible mechanisms by which SST become thin are discussed and could include changes in integrity of the suspensory apparatus with physiological events.

A prospective cohort study of digital cushion and corium thickness. Part 1: Associations with body condition, lesion incidence, and proximity to calving.

Claw horn disruption lesions (CHDL) are a major cause of lameness in dairy cattle and are likely a result of excessive forces being applied to the germinal epithelium that produces the claw horn. The digital cushion is a connective tissue structure, containing depots of adipose tissue, that sits beneath the distal phalanx and has been shown to be thicker in fatter cows. Body condition score (BCS) loss is a risk factor for CHDL, and one possible explanation is that fat is mobilized from the digital cushion during negative energy balance, causing the digital cushion to thin and lose force-dissipating capacity, leading to disruption of claw horn growth. This prospective cohort study investigated the association between measures of body fat and sole soft tissue (SST) thickness (a combined measure of the corium and digital cushion beneath the distal phalanx) in a longitudinal manner. The SST of 179 cows in 2 high-yielding dairy herds were measured at 5 assessment points between 8 wk before and 35 wk postcalving. The BCS, back fat thickness (BFT), and lesion incidence were recorded. Data were analyzed in a 4-level mixed effects regression model, with the outcome being SST thickness beneath the flexor tuberosity of the distal phalanx. Data from 827 assessment points were available for analysis. The overall mean of SST was 4.99 mm (standard deviation: 0.95). The SST was thickest 8 wk before calving (5.22 mm, standard deviation: 0.91) and thinnest 1 wk postcalving (4.68 mm, standard deviation: 0.87), suggesting an effect of calving on SST. The BFT was positively correlated with SST in the model with a small effect size (a 10 mm decrease in BFT corresponded with a 0.13 mm decrease in SST), yet the nadir of BFT was 11.0 mm at 9 to 17 wk postcalving (when SST was ∼4.95 mm), rather than occurring with the nadir of SST immediately after calving. The SST also varied with other variables [e.g., cows that developed a sole ulcer or severe sole hemorrhage during the study had thinner SST (-0.24 mm)], except when a sole ulcer was present, when it was thicker (+0.53 mm). Cows that developed lesions had a thinner digital cushion before the lesion occurrence, which became thickened with sole ulcer presence, perhaps representing inflammation. Furthermore, although BFT was correlated with SST over time, SST may also have been influenced by other factors such as integrity of the suspensory apparatus, which could have a major effect on CHDL. Measures of body fat likely contributed to having thin SST, but other factors including calving, herd, and lesion presence also had an effect.

Linking bone development on the caudal aspect of the distal phalanx with lameness during life.

Claw horn disruption lesions (CHDL; sole hemorrhage, sole ulcer, and white line disease) cause a large proportion of lameness in dairy cattle, yet their etiopathogenesis remains poorly understood. Untreated CHDL may be associated with damage to the internal anatomy of the foot, including to the caudal aspect of the distal phalanx upon which bone developments have been reported with age and with sole ulcers at slaughter. The primary aim of this study was to assess whether bone development was associated with poor locomotion and occurrence of CHDL during a cow's life. A retrospective cohort study imaged 282 hind claws from 72 Holstein-Friesian dairy cows culled from a research herd using X-ray micro-computed tomography (µ-CT; resolution: 0.11mm). Four measures of bone development were taken from the caudal aspect of each distal phalanx, in caudal, ventral, and dorsal directions, and combined within each claw. Cow-level variables were constructed to quantify the average bone development on all hind feet (BD-Ave) and bone development on the most severely affected claw (BD-Max). Weekly locomotion scores (1-5 scale) were available from first calving. The variables BD-Ave and BD-Max were used as outcomes in linear regression models; the explanatory variables included locomotion score during life, age, binary variables denoting lifetime occurrence of CHDL and of infectious causes of lameness, and other cow variables. Both BD-Max and BD-Ave increased with age, CHDL occurrence, and an increasing proportion of locomotion scores at which a cow was lame (score 4 or 5). The models estimated that BD-Max would be 9.8mm (SE 3.9) greater in cows that had been lame at >50% of scores within the 12mo before slaughter (compared with cows that had been assigned no lame scores during the same period), or 7.0mm (SE 2.2) greater if the cow had been treated for a CHDL during life (compared with cows that had not). Additionally, histology demonstrated that new bone development was osteoma, also termed "exostosis." Age explained much of the variation in bone development. The association between bone development and locomotion score during life is a novel finding, and bone development appears specific to CHDL. Bone development on the most severely affected foot was the best explained outcome and would seem most likely to influence locomotion score. To stop irreparable anatomical damage within the foot, early identification of CHDL and effective treatment could be critical.

Comparison of thermal imaging and rectal temperature in the diagnosis of pyrexia in pre-weaned calves using on farm conditions.

Measuring core body temperature is used as part of the diagnostic process in assessing the health of animals. Typically in calves, this is carried out using a rectal thermometer which can be time consuming, stressful to the calf and is invasive by nature. A non-invasive technique that is gaining recognition is thermal imaging. This study investigated the use of thermal imaging as a technique to assess core body temperature in pre-weaned artificially reared calves. A total of 125 male and female calves had rectal temperatures measured daily from day 7 until day 40 of life, and at the same time had a thermal image taken of the area around the medial canthus of the eye. A weak correlation (r = 0.28) was found between calf rectal temperature and thermal image temperature. A multivariable predictive model for core body temperature increased the correlation (r = 0.32) when including the environmental parameters of air temperature (p < .001) and wind speed (p < .001) as well as reconstituted milk replacer consumption (p < .01). The effectiveness of a predictive model including these parameters for the detection of calves with a core body temperature ≥ 39.5 °C was examined and found to have a sensitivity of 0% and a specificity of 100%. The results of this study demonstrate the need to take thermal environmental parameters into consideration when using thermal imaging to assess body temperature. However, the results suggest that accurate measures of core body temperature using thermal imaging cannot be achieved under commercial farm conditions. Further research is needed to determine what other factors could be measured to increase predictive ability.

S338 phosphorylation of Raf-1 is independent of phosphatidylinositol 3-kinase and Pak3.

The Raf-1 serine/threonine protein kinase requires phosphorylation of the serine at position 338 (S338) for activation. Ras is required to recruit Raf-1 to the plasma membrane, which is where S338 phosphorylation occurs. The recent suggestion that Pak3 could stimulate Raf-1 activity by directly phosphorylating S338 through a Ras/phosphatidylinositol 3-kinase (Pl3-K)/-Cdc42-dependent pathway has attracted much attention. Using a phospho-specific antibody to S338, we have reexamined this model. Using LY294002 and wortmannin, inhibitors of Pl3-K, we find that growth factor-mediated S338 phosphorylation still occurs, even when Pl3-K activity is completely blocked. Although high concentrations of LY294002 and wortmannin did suppress S338 phosphorylation, they also suppressed Ras activation. Additionally, we show that Pak3 is not activated under conditions where S338 is phosphorylated, but when Pak3 is strongly activated, by coexpression with V12Cdc42 or by mutations that make it independent of Cdc42, it did stimulate S338 phosphorylation. However, this occurred in the cytosol and did not stimulate Raf-1 kinase activity. The inability of Pak3 to activate Raf-1 was not due to an inability to stimulate phosphorylation of the tyrosine at position 341 but may be due to its inability to recruit Raf-1 to the plasma membrane. Taken together, our data show that growth factor-stimulated Raf-1 activity is independent of Pl3-K activity and argue against Pak3 being a physiological mediator of S338 phosphorylation in growth factor-stimulated cells.

Randomised controlled trial to evaluate the effect of foot trimming before and after first calving on subsequent lameness episodes and productivity in dairy heifers.

The objective of this study was to assess both independent and combined effects of routine foot trimming of heifers at 3 weeks pre-calving and 100 days post calving on the first lactation lameness and lactation productivity. A total of 419 pre-calving dairy heifers were recruited from one heifer rearing operation over a 10-month period. Heifers were randomly allocated into one of four foot trimming regimens; pre-calving foot trim and post-calving lameness score (Group TL), pre-calving lameness score and post-calving foot trim (Group LT), pre-calving foot trim and post-calving foot trim (Group TT), and pre-calving lameness score and post-calving lameness score (Group LL, control group). All heifers were scored for lameness at 24 biweekly time points for 1 year following calving, and first lactation milk production data were collected. Following calving, 172/419 (41.1%) of heifers became lame during the study (period prevalence), with lameness prevalence at each time-point following calving ranging from 48/392 (12.2%) at 29-42 days post-calving to 4/379 (1.1%) between 295 and 383 days after calving. The effects of the four treatment groups were not significantly different from each other for overall lameness period prevalence, biweekly lameness point prevalence, time to first lameness event, type of foot lesion identified at dry off claw trimming, or the 4% fat corrected 305-day milk yield. However, increased odds lameness was significantly associated with a pre-calving trim alone (P = 0.044) compared to the reference group LL. The odds of heifer lameness were highest between 0 and 6 weeks post-partum, and heifer farm destination was significantly associated with lameness (OR 2.24), suggesting that even at high standard facilities, environment and management systems have more effect on heifer foot health than trimming.

Isolation of a novel RXR from Xenopus that most closely resembles mammalian RXR beta and is expressed throughout early development.

In a search for nuclear receptors that may mediate the teratogenic effects of the potential morphogen, retinoic acid, on the early development of Xenopus we have isolated a novel Xenopus RXR that most closely resembles the mammalian beta-type RXR. Xenopus RXR beta mRNA is expressed throughout early embryogenesis, and functions as an accessory protein to enhance the DNA-binding of other members of the nuclear receptor superfamily.

Phase I study of intrapleural batimastat (BB-94), a matrix metalloproteinase inhibitor, in the treatment of malignant pleural effusions.

Tumor cells and associated stromal cells secrete matrix metalloproteinases (MMPs), contributing to invasion, angiogenesis, and metastasis. Batimastat (BB-94) is a broad-spectrum MMP inhibitor that causes resolution of ascites and/or tumor growth delay in animal models of breast, ovarian, and colorectal cancer. We recruited 18 patients with cytologically positive malignant pleural effusions into a Phase I study of intrapleural BB-94. Three patients received single doses of BB-94 at each dose level: 15, 30, 60, 105, 135, and 300 mg/m2. Two patients were retreated with a second course at 60 and 105 mg/m2. BB-94 was detectable in plasma 1 h after intrapleural administration, and peak levels of 20-200 ng/ml occurred after 4 h to 1 week. BB-94 persisted in the plasma for up to 12 weeks, at levels exceeding the IC50s for target MMPs. Peak values were higher, and persistence in the plasma was longer after higher doses of BB-94. The treatment was well tolerated. Toxic effects included low-grade fever for 24-48 h (6 of 18 patients, 33%) and reversible asymptomatic elevation of liver enzymes (8 patients, 44%). Toxicity seemed unrelated to BB-94 dose or plasma levels. Sixteen patients evaluable for response required significantly fewer pleural aspirations in the 3 months after BB-94 compared with the 3 months before. Seven patients (44%) required no further pleural aspiration until death/last follow-up. After 1 month, patients treated with 60-300 mg/m2 BB-94 had significantly better dyspnea scores, indicating improved exercise tolerance, compared with baseline scores the day after BB-94. The maximum tolerated intrapleural dose remains to be defined, but it is clear that intrapleural BB-94 is well tolerated, with evidence of local activity.

Claw length recommendations for dairy cow foot trimming.

The aim was to describe variation in length of the dorsal hoof wall in contact with the dermis for cows on a single farm, and hence, derive minimum appropriate claw lengths for routine foot trimming. The hind feet of 68 Holstein-Friesian dairy cows were collected post mortem, and the internal structures were visualised using x-ray µCT. The internal distance from the proximal limit of the wall horn to the distal tip of the dermis was measured from cross-sectional sagittal images. A constant was added to allow for a minimum sole thickness of 5 mm and an average wall thickness of 8 mm. Data were evaluated using descriptive statistics and two-level linear regression models with claw nested within cow. Based on 219 claws, the recommended dorsal wall length from the proximal limit of hoof horn was up to 90 mm for 96 per cent of claws, and the median value was 83 mm. Dorsal wall length increased by 1 mm per year of age, yet 85 per cent of the null model variance remained unexplained. Overtrimming can have severe consequences; the authors propose that the minimum recommended claw length stated in training materials for all Holstein-Friesian cows should be increased to 90 mm.

Effects of retinoic acid on Xenopus embryos.

Retinoic acid (RA) has powerful dose-dependent dysmorphogenic effects on Xenopus embryos. The defects produced are dependent upon the stage and duration of treatment. Characteristic dysmorphogenic effects occur in the visceral (branchial) arch region and the tail. These regions correspond to the domains of expression of the retinoid receptors RAR gamma and RXR beta. By expressing domain-swapped and dominant negative derivatives of RAR gamma in embryos, we have shown that the effects of RA are mediated transcriptionally. Furthermore, the expression of dominant negative receptors in the absence of exogenous ligand has no apparent harmful effect upon early development. Finally, we have identified a novel MAP kinase phosphatase whose expression pattern is localized to regions similar to RAR gamma, and which is upregulated by RA.

Bronchopneumonia in mice caused by Pasteurella haemolytica A2 after predisposition by ovine Bordetella parapertussis.

Initial intranasal inoculation of four to eight-week-old Swiss White mice with 7.5 x 10(6) colony forming units (cfu) of ovine B. parapertussis followed 30 min, three or five days, by intranasal inoculation with 1.4 x 10(5) cfu of Pasteurella haemolytica A2 resulted in a more severe infection pattern than when either agent was administered alone. Histopathological examination showed that inoculation with B. parapertussis alone caused a bronchopneumonia the severity of which was dependant upon the infecting dose. Bacteria were recovered up to 10 days after inoculation. P. haemolytica alone had no apparant pathogenic effect and was cleared from the lungs within 24 h. When both agents were given in combination the lesions were most severe when P. haemolytica was administered three days after B. parapertussis infection. These findings suggest that B. parapertussis predisposes mice to subsequent infection with P. haemolytica and that the timing of the P. haemolytica administration influences the severity of the lung lesions.

A survey of the on-farm treatment of sole ulcer and white line disease in dairy cattle.

A telephone survey of UK dairy farmers was conducted to investigate current on-farm practice in the treatment of mild sole ulcer (SU)/sole bruising (SB), and white line disease (WLD), and the potential barriers associated with therapy. A total of 84 dairy farmers were questioned about the process of detecting and treating lame cows on their farm as well as about the specific treatments they applied. Farmers were also canvassed for their views on the efficacy of different potential treatments for mild SU/SB and WLD. In general, respondents discussed treatments for SU and WLD rather than specifically for mild SU/SB and WLD. Furthermore, when describing treatment methods, farmers rarely differentiated between SU and WLD. Trimming the affected claw with or without the additional use of orthopaedic blocks was the most commonly reported treatment method considered effective and practical by the majority of farmers. Antibiotics and/or analgesics were used by a small number of farmers, and some housed their most severely lame cows in straw pens. Lack of time, inadequate equipment and poor farm layout were identified by many survey respondents as barriers to the prompt and/or effective treatment of their lame animals.

A novel nuclear receptor superfamily member in Xenopus that associates with RXR, and shares extensive sequence similarity to the mammalian vitamin D3 receptor.

We report the isolation of xONR1, a novel member of the nuclear receptor superfamily from Xenopus laevis. xONR1 shares a high degree of amino acid sequence identity with the mammalian receptor for 1 alpha, 25-dihydroxy vitamin D3, particularly within the DNA-binding domain, although it does not bind this ligand. xONR1 DNA binding is stimulated by association with retinoid X receptor gamma (RXR gamma).

Mesoderm induction in Xenopus caused by activation of MAP kinase.

Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras (ref. 5). Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction, and the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. Here we show that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation.

Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases.

It has previously been shown that maximal activation of Raf-1 is produced by synergistic signals from oncogenic Ras and activated tyrosine kinases. This synergy arises because Ras-GTP translocates Raf-1 to the plasma membrane where it becomes phosphorylated on tyrosine residues 340 and 341 by membrane-bound tyrosine kinases (Marais, R., Light, Y., Paterson, H. F., and Marshall, C. J. (1995) EMBO J. 14, 3136-3145). We have examined whether the other two members of the Raf family, A-Raf and B-Raf, are regulated in a similar way to Raf-1. A-Raf behaves like Raf-1, being weakly activated by oncogenic Ras more strongly activated by oncogenic Src, and these signals synergize to give maximal activation. B-Raf by contrast is strongly activated by oncogenic Ras alone and is not activated by oncogenic Src. These results show that maximal activation of B-Raf merely requires signals that generate Ras-GTP, whereas activation of Raf-1 and A-Raf requires Ras-GTP together with signals that lead to their tyrosine phosphorylation. B-Raf may therefore be the primary target of oncogenic Ras.