Synthesis of proteins by automated flow chemistry.

Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

Clinical features, intestinal histopathology, and outcome in protein-losing enteropathy in Yorkshire Terrier dogs.

BACKGROUND: A poorly understood protein-losing enteropathy (PLE) disorder has been reported in Yorkshire Terrier dogs. OBJECTIVES: To describe clinical features, intestinal histopathology, and outcome in Yorkshire Terrier dogs with PLE, and to identify variables predictive of outcome. ANIMALS: Thirty client-owned Yorkshire Terrier dogs with PLE. METHODS: Retrospective study. Records of dogs with a diagnosis of PLE were reviewed. Intestinal histopathology was interpreted using the World Small Animal Veterinary Association gastrointestinal histopathology classification system. Discriminate analysis techniques were used to identify variables predictive of outcome. RESULTS: Females outnumbered males (20/30). Median age was 7 years (range 1-12). Common clinical signs were diarrhea (20/30), vomiting (11), ascites and abdominal distension (11), and respiratory difficulty (8). Histopathologic abnormalities included villous lymphatic dilatation, crypt lesions, villous stunting, and variable increases in cellularity of the lamina propria. All dogs were treated with glucocorticoids. Of 23 dogs with long-term follow-up, 9 had complete, and 3 had partial, resolution of signs, and 11 failed to respond to treatment. Median survival of responders was 44 months and of nonresponders was 12 months, with 4 dogs experiencing peracute death. Vomiting, monocytosis, severity of hypoalbuminemia, low blood urea nitrogen concentration, and villous blunting were predictive of survival <4 months. CONCLUSIONS: In addition to classic GI signs, Yorkshire Terriers with PLE often show clinical signs associated with hypoalbuminemia and low oncotic pressure. Lymphatic dilatation, crypt lesions, and villous stunting are consistent histopathologic findings. Clinical outcomes are variable, but many dogs experience remission of clinical signs and prolonged survival.

Applicability of 3T body MRI in assessment of nonfocal bone marrow involvement of hematopoietic neoplasia in dogs.

BACKGROUND: The utility of whole body magnetic resonance imaging (MRI) in detecting bone marrow infiltration in dogs with cancer has not been investigated. OBJECTIVES: To assess the feasibility of 3T body MRI for bone marrow assessment in dogs with hematopoietic neoplasia. ANIMALS: Seven dogs with B-cell lymphoma, 3 dogs with myelodysplastic syndrome (MDS), and 2 clinically normal dogs. METHODS: A prospective study of dogs with hematopoetic cancer was conducted using T1W, T2W, In-Phase, Out-of-Phase and STIR pulse sequences of the body excluding the head prior to bone marrow sampling. The relative signal intensity of a midlumbar vertebral body and a midshaft femoral bone marrow was compared by visual and point region of interest analysis to regional skeletal muscle. RESULTS: Similarity of femoral diaphyseal and vertebral body marrow signal intensity to that of skeletal muscle on the Out-of-Phase sequence was useful in distinguishing the 3 dogs with hypercellular marrow because of MDS from the 7 dogs with B-cell lymphoma and from the 2 clinically normal dogs. 1/7 dogs with lymphoma had proven bone marrow involvement but normal cellularity and less than 5% abnormal cells. Unaffected midfemoral marrow had greater signal intensity than skeletal muscle and unaffected vertebral marrow had less signal intensity than skeletal muscle on the Out-of-Phase sequence. CONCLUSIONS AND CLINICAL IMPORTANCE: 3T, Out-of-Phase MR pulse sequence was useful in distinguishing diffuse bone marrow infiltrate (MDS) from minimally or unaffected marrow using skeletal muscle for signal intensity comparison on whole body MRI.

Life-threatening systemic toxicity and airway compromise from a common European adder bite to the tongue.

A 24-year-old man was bit on the tongue by a European common adder. Within 15 min following envenomation, he experienced tongue swelling, hypotension and impaired consciousness. Antihistamine, corticosteroid and crystalloids were administered. Within 105 min of envenomation, increasing oral, pharyngeal and facial oedema compromised the airway, leading to respiratory failure, concomitant with circulatory failure related to hypoxaemia and systemic toxic effects. Acute tracheotomy secured the airway, and two doses of antivenom successfully treated the systemic, toxic effects. The reaction was severe due to rapid and suspected high-dose uptake of venom, underlining the need for early advanced symptomatic treatment with airway control and early and eventually repeated dosing of antivenom.

External-beam Co-60 radiotherapy for canine nasal tumors: a comparison of survival by treatment protocol.

A retrospective analysis of survival times in dogs with intranasal tumors was performed comparing those treated using hypofractionated or full course Co-60 radiotherapy protocols alone or with surgical adjuvant therapy and those receiving no radiation treatment. One hundred thirty-nine dogs presented to the University of Minnesota Veterinary Medical Center for treatment of histologically-confirmed nasal neoplasia between July 1983 and October 2001 met the criteria for review. Statistically analyzed parameters included age at diagnosis, tumor histologic classification, fractionation schedule (number of treatments, and number of treatment days/week) (classified as hypofractionated if 2 or less treatments/week); calculated minimum tumor dose/fraction; calculated total minimum tumor dose (classified as hypofractionated if less than 37 Gy in six or fewer fractions); number of radiotherapy portals, a treatment gap of more than 7 days in a full course (3-5 treatments/week, 3-3.5 week treatment time) radiotherapy protocol, the influence of eye shields on survival following single portal DV fields, the survey radiographic extent of the disease, and the presence or absence of cytoreductive surgery. There was a significant relationship only between protocols using 3 or more treatments/week and at least 37 Gy cumulative minimum tumor dose and survival. However, there was no significant relationship between either total minimum tumor dose or dose/fraction and survival and there were no significant relationships between survival and any of the other variables analyzed including tumor histologic type.

Selective brain cooling in mammals and birds.

Artiodactyls and felids have a carotid rete that can cool the blood destined for the brain and consequently the brain itself if the cavernous sinus receives cool blood returning from the nose. This condition is usually fulfilled in resting and moderately hyperthermic animals. During severe exercise hyperthermia, however, the venous return from the nose bypasses the cavernous sinus so that brain cooling is suppressed. This is irreconcilable with the assumption that the purpose of selective brain cooling (SBC) is to protect the brain from thermal damage. Alternatively, SBC is seen as a mechanism engaging the thermoregulatory system in a water-saving economy mode in which evaporative heat loss is inhibited by the effects of SBC on brain temperature sensors. In nonhuman mammals that do not have a carotid rete, no evidence exists of whole-brain cooling. However, the surface of the cavernous sinus is in close contact with the base of the brain and is the likely source of unregulated regional cooling of the rostral brain stem in some species. In humans, the cortical regions next to the inner surface of the cranium are very likely to receive some regional cooling via the scalp-sinus pathway, and the rostral base of the brain can be cooled by conduction to the nearby respiratory tract; mechanisms capable of cooling the brain as a whole have not been found. Studies using conventional laboratory techniques suggest that SBC exists in birds and is determined by the physical conditions of heat transfer from the head to the environment instead of physiological control mechanisms. Thus except for species possessing a carotid rete, neither a coherent pattern of SBC nor a unifying concept of its biological significance in mammals and birds has evolved.

An integrated epidemiologic and radiographic algorithm for canine urocystolith mineral type prediction.

Research involved 2 databases. One database (occurrence frequency) comprised the age, breed, gender and urocystolith mineral type (pure chemical types only) from 2041 canine patients submitted to the Minnesota Urolith Center. The other database (imaging) comprised the maximum size, surface (rough, smooth, and smooth with blunt tips), shape (faceted, irregular, jackstone, ovoid, and round) and internal architecture (lucent center, random-nonuniform, and uniform) from 434 canine patients imaged in a urinary bladder phantom. The imaging database was a partial subset of the occurrence frequency database. Imaging techniques simulated were survey radiography and double contrast cystography. The databases were compared using multivariate analysis techniques. Equations were developed to use clinically-relevant characteristics (age, breed, gender, maximum size, surface, shape, and internal architecture) to predict urocystolith mineral types. The goal was to assess the accuracy of the various techniques in predicting the urocystolith mineral types. The combination of signalment (age, breed, gender) and simulated survey radiographic findings does not improve mineral type prediction accuracy (average across all mineral types is 69.9%) beyond that achievable with signalment alone (average across all mineral types is 69.8%). However, the combination of signalment and double contrast cystography does improve mineral type prediction accuracy (average across all mineral types is 75.3%). For comparison, mineral type prediction accuracy without signalment from survey radiographs only was 65.7% across all mineral types. The clinical utility of the algorithm is the option to distinguish urocystolith mineral types requiring surgical vs. medical treatment.

Relevance of sonographic artifacts observed during in vitro characterization of urocystolith mineral composition.

Nine pure mineral type canine uroliths (bladder or urethral origin only) were imaged ultrasonographically using 3.5 MHz, 5.0 MHz, and 7.5 MHz fixed focus, mechanical sector transducers in a urinary bladder phantom. The uroliths studied were those composed of 100% magnesium ammonium phosphate, calcium oxalate monohydrate, calcium oxalate dihydrate, calcium phosphate appatite, and calcium hydrogen phosphate dihydrate (brushite), ammonium acid urate, sodium acid urate, cystine, and silica. The occurrence of both reverberation/streak and acoustic shadowing artifacts were compared to urocystolith mineral type (classified by effective atomic number), urocystolith width, urocystolith height (thickness), and ultrasonographic imaging frequency. No predictable relationship was found between either of the artifacts seen beyond the urocystolith (reverberation/streak or the acoustic shadowing) and urocystolith mineral type. There was no statistical relationship between the occurrence of reverberation/streak artifact and the size (width or height) of the urocystolith or the ultrasonographic frequency. There was, however, a statistically relevant relationship between ultrasonographic imaging frequency and the occurrence of acoustic shadowing and between urocystolith height (thickness) and the occurrence of acoustic shadowing. However, regardless of ultrasound frequency, acoustic shadowing was observed less than 35% of the time in any of the urocystolith mineral types examined. Based on the imaging of the bladder phantom supporting apparatus (7.0 mm bolts covered by plastic), the accurate characterization of a curved object surface directly facing the transducer was found to be directly related to the frequency of sound used for imaging and at best predictably limited to curved vs flat. Accurate measurement of the maximum transverse dimension of an echogenic curved object or accurate characterization of the lateral borders of such an object was considered unlikely with general ultrasonographic equipment of the frequencies studied. Therefore, detailed architectural characterization of urocystoliths suitable for mineral composition prediction is considered highly unlikely with general pulse-echo ultrasonographic techniques.

Measurement of health status and quality of life in neonatal follow-up studies.

Mortality and neuro-developmental outcome can be precisely measured but in order to interpret the significance of changes in them, further information is needed about health status, the effect of health status on lifestyle of survivors and their families, the quality of life experienced by survivors and the value placed by the public and survivors on different health states. The ability to measure such aspects of health now allows more relevant follow-up studies to be designed. This article discusses concepts of health status and quality of life and the problems in applying these to children. There follows a critique of modern instruments for measuring health status and quality of life and their application in neonatal follow-up studies. We recommend that only a small number of well-established instruments should be considered so that results are valid and can be compared with other studies.

Contrast medium-related artifacts observed during in vitro radiographic characterization of urocystolith mineral composition.

Nine pure mineral types of canine uroliths (bladder or urethral origin only) were exposed to sequential increasing concentrations of iodinated, radiographic contrast medium in petri dishes. The uroliths studied were those composed of 100% magnesium ammonium phosphate, calcium oxalate monohydrate, calcium oxalate dihydrate, calcium phosphate appatite, and calcium hydrogen phosphate dihydrate (Brushite), ammonium acid urate, sodium acid urate, cystine, and silica. Two phenomena were observed. First, there was a tendency for selected urocystoliths to undergo radiopacity augmentation beyond that expected for just contrast medium superimposition. This was termed, contrast medium adhesion, which persisted despite repeated washing of the urocystoliths. Second, there was a tendency for bubbles to form on or near selected urocystolith chemical types. These observations prompted careful scrutiny for their occurrence in subsequent clinical simulation of radiographic procedures using these same urocystoliths in a urinary bladder phantom. Imaging techniques simulated were survey radiography, pneumocystography, double contrast cystography (two iodine concentrations). The contrast medium adhesion occurrence found in the petri dish studies was compared to urocystolith mineral type. Similar comparisons were made for contrast medium adhesion occurrence in the bladder phantom. The detection of contrast medium adhesion in the bladder phantom differed from that observed in the petri dish experiments. While contrast adhesion occurred across a fairly broad range of the urocystolith mineral types in the petri dish studies, it was observed primarily for sodium acid urate and cystine urocystoliths in the bladder phantom. Prompted by the observation of bubbles in association with a limited number of urocystolith types in the petri dish studies, bubble occurrence in the bladder phantom was compared to the urocystolith type. Bubble formation on or near the urocystoliths, although uncommonly observed, was seen only with either cystine or silica urocystoliths. The potential clinical utility and clinical caveat aspects of these phenomena are discussed.

Loss of urocystolith architectural clarity during in vivo radiographic simulation versus in vitro visualization.

Urocystoliths of 9 mineral types from 434 canine patients submitted to the University of Minnesota Urolith Bank were imaged in a urinary bladder phantom. Imaging techniques simulated were survey radiography and double contrast cystography. Morphologic characteristics visually observed in vitro or by interpretation of high-resolution specimen radiographs were compared to those seen using the simulated in vivo imaging techniques. Shape characteristics that were accurately detected > or = 25% of the time on simulated survey or double contrast radiography were faceted, irregular, jackstone, ovoid, and round. Surface characteristics that were accurately detected > or = 25% of the time on simulated survey or double contrast radiography were rough, smooth, and smooth with blunt tips. Internal architecture characteristics that were accurately detected > or = 25% of the time on simulated survey or double contrast radiography were lucent center, random-nonuniform, and uniform. Shapes such as bosselated, faceted-ovoid, and rosette; surfaces such as botryoidal, and knife-edged; and internal architecture characteristics such as dense center, dense shell, laminated, and fissures were of almost no value either due to poor detectability or poor accuracy of recognition. Based on optimized simulated survey and double contrast radiographic procedures, it appears that a number of shape, surface, and internal architecture characteristics may be of limited or no value in discriminating among urocystolith mineral types under clinical circumstances. Shapes and surfaces were more accurately characterized by the simulated double contrast technique, but for internal architecture, the simulated survey radiographic technique seemed slightly superior overall.

Urocystolith detection: comparison of survey, contrast radiographic and ultrasonographic techniques in an in vitro bladder phantom.

Urocystoliths of 9 mineral types from 437 canine patients submitted to the University of Minnesota Urolith Bank were imaged in a urinary bladder phantom. Imaging techniques simulated were survey radiography, pneumocystography, double contrast cystography (two iodine concentrations) and real-time ultrasonography (3.5 MHz, 5.0 MHz, 7.5 MHz). Imaging techniques were compared for accuracy of urocystolith detection, accuracy of urocystolith enumeration, and tendencies for over or undercounting. Across urocystolith mineral types, the false negative rates (no urocystoliths detected in a given case) for survey radiographs range from 2 to 27%. Pneumocystographic techniques are one-half as likely to yield false negative results as are survey radiographic techniques. Underestimates of urocystolith numbers and false negatives are likely using 80 mg iodine/ml double contrast cystography because calcium-based urocystoliths are isopaque in this contrast medium dilution. The 200 mg iodine/ml double contrast cystographic techniques are unlikely to yield false negative diagnoses even for very small (< or = 1.0 mm) urocystoliths and is comparable to pneumocystography for detection and slightly better for enumeration. The likelihood of an ultrasonographic false negative for urocystoliths decreases with increasing MHz. Under optimal conditions using a 7.5 MHz mechanical sector transducer, the false negative rates were comparable to double contrast cystography, but rates increased notably with lower MHz transducers.

Imaging canine urocystoliths. Detection and prediction of mineral content.

The role of survey radiography, contrast radiography, and ultrasonography in the assessment of urocystolithiasis has been primarily one of detection. Information about the radiographic characteristics of uroliths (size, shape, radiopacity, surface characteristics, number detected) for characterization and diagnosis of urocystolithiasis is emerging; however, there are no studies on the accuracy or sensitivity of these techniques. In this article, the intent is to update knowledge about available imaging techniques used in the detection and characterization of urocystoliths based on recent studies designed to evaluate their accuracy in prediction of different urocystoliths mineral content.

In vitro evaluation of contrast medium concentration and depth effects on the radiographic appearance of specific canine urolith mineral types.

Nine pure mineral types of canine uroliths (bladder or urethral origin only) identified in a chronologic sample from the Minnesota Urolith Center were compared to sequential dilutions of iodinated radiographic contrast medium in vitro. The uroliths studied were those composed of 100% magnesium ammonium phosphate, calcium oxalate monohydrate, calcium oxalate dihydrate, calcium phosphate appatite, calcium hydrogen phosphate dihydrate (brushite), ammonium acid urate, sodium acid urate, cystine, and silica. The radiopacity of the uroliths was classified as radiolucent, isopaque, or radiopaque, as compared to the radiopacity of the contrast medium solutions in which they were placed, using 2.0 mm and 5.0 mm depths in petri dishes radiographed using a table-top technique. A statistically significant relationship was found between the effective atomic number of the uroliths and the effective atomic number of the contrast medium solutions to which they were compared for the endpoints of isopacity, first lucency (in increasing iodine concentration sequence), and optimal visualization of internal architecture. In general, uroliths isopaque or radiolucent in contrast medium solutions weaker than 23.5 mgI2/ml are most likely ammonium acid urate or sodium acid urate. Uroliths isopaque or radiolucent in contrast medium solutions between 23.5 mgI2/ml and 44.4 mgI2/ml are probably magnesium ammonium phosphate, cystine, or silica. Uroliths that remained radiopaque in solutions stronger than 44.4 mgI2/ml, and particularly those radiopaque in contrast medium solutions stronger than 80 mgI2/ml, almost always contained calcium. This relative opacity assessment is proposed for use in double contrast cystography as an aid in differentiating urolith mineral types clinically to facilitate appropriate use of medical protocols to dissolve uroliths or to prevent their growth or recurrence.

Effects of dehydration and rehydration on body temperatures in the black bedouin goat.

The temperatures of the arterial blood and the brain in black Bedouin goats were measured continuously by miniature data loggers. The animals were either euhydrated or dehydrated to 75-80% of the initial body mass by withholding water for 3-4 days during exposure to intense solar radiation. The daily blood temperature means and maxima of were significantly higher in dehydration than in euhydration, but 40 degreesC was rarely exceeded even during the hot hours of the day. Selective brain cooling occurred in euhydration, but its extent was small when blood temperature was below 39.5 degreesC. In dehydration, however, selective brain cooling was frequent and the standard response when blood temperature exceeded 39 degreesC. We believe that selective brain cooling contributes to the inhibition of evaporative heat loss, which is the primary cause of the higher blood temperature in dehydration. Rapid rehydration with cold water induced long-lasting depression of blood temperature. No evidence was found for mechanisms attenuating the subsequent decrease of brain temperature which occurred a few minutes after the uptake of cold water.

Evaluation of the morphologic characteristics and prevalence of canine urocystoliths from a regional urolith center.

OBJECTIVE: To determine whether morphology of single-mineral urocystoliths and age, sex, or breed data could be applied to facilitate radiographic and clinical urocystolith mineral type prediction, respectively, in dogs. SAMPLE POPULATION: Database of 2,041 dogs with pure mineral composition urocystoliths. PROCEDURE: All uroliths were characterized according to geologic descriptive terminology and by breed, sex, and age of dog at time of sample submission. Summary statistics were used to compare features with specific mineral types. Observed trends were analyzed for statistical relevance between observed and expected frequencies for age, sex, color, size, shape, and surface, using the null hypothesis that differences by urocystolith mineral type did not exist. On the basis of expected breed occurrence derived by equations, the null hypothesis that urocystolith occurrence paralleled canine breed popularity was tested. RESULTS: Urocystoliths > 10 mm in any dimension were > 92% likely to be magnesium ammonium phosphate hexahydrate (MAP). Smooth, blunt-edged or faceted, and pyramidal urocystoliths were usually MAP. Jackstone shapes were almost always silica. Botryoidal (grape-like clusters) urocystoliths were likely to be oxalates. Breeds with high relative likelihood of urocystoliths included: English Bulldog, Pekingese, Pug, Welsh Corgi, and West Highland White Terrier. Breeds with low relative likelihood of urocystolith production included: German Shepherd Dog, Shar-Pei, and German Shorthaired Pointer. About 94% of urocystoliths produced in females or spayed females were MAP, whereas males and neutered males produced a greater assortment. CONCLUSIONS AND CLINICAL RELEVANCE: For pure mineral composition urocystoliths, trends in mineral type among breeds and between sexes can be exploited clinically in the diagnosis and management of urolith-related disease. Size and shape, used in conjunction with age, breed, and sex, can facilitate pure urocystolith mineral type prediction.

Activity, blood temperature and brain temperature of free-ranging springbok.

We used miniature data loggers to record temperature and activity in free-ranging springbok (Antidorcas marsupialis) naturally exposed to severe nocturnal cold and moderate diurnal heat. The animals were active throughout the day and night, with short rests; the intensity of activity increased during daylight. Arterial blood temperature, averaged over many days, exhibited a circadian rhythm with amplitude < 1 degree C, but with a wide range which resulted from sporadic rapid deviations of body temperature. Peak blood temperature occurred after sunset. Environmental thermal loads had no detectable effect on blood temperature, even though globe temperature varied by > 10 degrees from day to day and > 20 degrees C within a day. Brain temperature increased approximately linearly with blood temperature but with a slope < 1, so that selective brain cooling tended to be activated at high body temperature, but without a precise threshold for the onset of brain cooling. Low activity attenuated selective brain cooling and high activity abolished it, even at high brain temperature. Our results support the concept that selective brain cooling serves to modulate thermoregulation rather than to protect the brain against heat injury.

Blood and brain temperatures of free-ranging black wildebeest in their natural environment.

Using miniature data loggers, we measured the temperatures of carotid blood and brain in four wildebeest (Connochaetes gnou) every 2 min for 3 wk and every 5 min, in two of the animals, for a further 6 wk. The animals ranged freely in their natural habitat, in which there was no shelter. They were subject to intense radiant heat (maximum approximately 1,000 W/m2) during the day. Arterial blood temperature showed a circadian rhythm with low amplitude (< 1 degree C) and peaked in early evening. Brain temperature was usually within 0.2 degrees C of arterial blood temperature. Above a threshold between 38.8 and 39.2 degrees C, brain temperature tended to plateau so that the animals exhibited selective brain cooling. However, selective brain cooling sometimes was absent even when blood temperature was high and present when it was low. During helicopter chases, selective brain cooling was absent, even though brain temperature was near 42 degrees C. We believe that selective brain cooling is controlled by brain temperature but is modulated by sympathetic nervous system status. In particular, selective brain cooling may be abolished by high sympathetic activity even at high brain temperatures.